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Red orange and lemon extract (RLE) is an anthocyanins-rich dietary supplement that may influence gastrointestinal bacterial community in ruminants. The aim of the present study was to investigate the RLE effects on gut microbiota composition in lambs. Twenty-eight lambs were randomly divided into a control group (CON, n = 14) and an anthocyanin group (ANT, n = 14) and fed the same diet, additionally, only the ANT received 90 mg/kg live weight of RLE at day. After lamb slaughter (40 ± 1 days), fecal samples were collected from the rectum and stored at −20 °C until analysis. Analysis of fecal microbiome was carried out by metabarcoding analysis of 16S rRNA. After reads denoising, sequences were aligned against SILVA rRNA sequence database using MALT, and taxonomic binning was performed with MEGAN. A significant increase in Firmicutes and Bacteroidetes and a decrease in Proteobacteria and Actinobacteria was observed in ANT compared to CON. Moreover, an interesting increase of Lactobacillus and Bifidobacterium genera and a decrease in Escherichia&nbsp, coli and Salmonella species were detected in ANT compared to CON. Results recommend that anthocyanin supplementation in lamb diet is able to modulate positively gut microbiota and may inhibit the growth of some potential pathogenic microorganisms.

Lactic acid bacteria (LAB) decisively influence the technological, nutritional, organoleptic and preservation properties of bakery products. Therefore, their use has long been considered an excellent strategy to improve the characteristics of those goods. The aim of this study was the evaluation of microbial diversity in different doughs used for the production of a typical Apulian flatbread, named focaccia. Leavening of the analyzed doughs was obtained with baker’s yeast or by applying an innovative “yeast-free” protocol based on a liquid sourdough obtained by using Leuconostoc citreum strain C2.27 as a starter. The microbial populations of the doughs were studied by both a culture-dependent approach and metagenetic analyses. The flours used for dough preparation were also subjected to the same analyses. The metagenetic analyses were performed by sequencing the V5–V6 hypervariable regions of the 16S rRNA gene and the V9 hypervariable region of the 18S rRNA gene. The results indicate that these hypervariable regions were suitable for studying the microbiota of doughs, highlighting a significant difference between the microbial community of focaccia dough with baker’s yeast and that of the dough inoculated with the bacterial starter. In particular, the dough made with baker’s yeast contained a microbiota with a high abundance of Proteobacteria (82% of the bacterial population), known to be negatively correlated with the biochemical properties of the doughs, while the Proteobacteria in dough produced with the L. citreum starter were about 43.5% lower than those in flour and dough prepared using baker’s yeast. Moreover, the results show that the L. citreum C2.27 starter was able to dominate the microbial environment and also reveal the absence of the genus Saccharomyces in the dough used for the production of the “yeast-free” focaccia. This result is particularly important because it highlights the suitability of the starter strain for obtaining an innovative “yeast-free” product.

Background: Due to coronavirus disease-19 (COVID-19) pandemic, Italian outpatient clinics were suspended in March-April 2020 and subsequently slowed down. Telemedicine was shown to be useful in headache clinics, despite absence of a detailed protocol for its development. Objective: To describe the implementation of a structured telemedicine protocol during COVID-19 pandemic. Materials and Methods: Since May 2020, we performed a quality improvement study in a Headache Specialist Center in central Italy. We involved patients who had in-person follow-up visits scheduled during suspension and initial reopening of clinics. Patients had two appointments with a nurse specialized in headache care and a headache physician, respectively, using Microsoft Teams®. The service is still active. We collected sociodemographic and clinical characteristics of patients, technical details of telemedicine visits, patient feedback, medical judgment about complexity of clinical decisions, and need for in-person re-evaluation. We also performed a Strengths-Weaknesses-Opportunities-Threats analysis to provide a realistic picture of the service. Results: We performed 207 telemedicine visits involving 100 patients with a median age of 44 (interquartile range [IQR]: 35-56) years; 76.0% were women and lived at a median of 68 (IQR: 24-109) km from the Center. Thirty-nine (39.0%) were visited for migraine without aura. Patients mostly used a computer (68.1% visits) with high audio-video quality in 93.2% of visits. First and second appointments lasted in median 20 (IQR: 14-25) minutes and 9 (IQR: 7-13) minutes, respectively. Interacting with patients was very easy in 66.7% of visits. Patients reported no difficulty in sharing documents and high satisfaction in 78.6% and 93.5% of visits, respectively. Perceived complexity of clinical decisions was generally low (86.5%), whereas 8.2% of cases required in-person re-evaluation. Conclusions: Telemedicine facilitated follow-ups, ensuring multidisciplinary care and high patient satisfaction, justifying its wider adoption in headache care.

Toxigenic fungi and mycotoxins are very common in food crops, with noticeable differences in their host specificity in terms of pathogenicity and toxin contamination. In addition, such crops may be infected with mixtures of mycotoxigenic fungi, resulting in multi-mycotoxin contamination. Climate represents the key factor in driving the fungal community structure and mycotoxin contamination levels pre- and post-harvest. Thus, there is significant interest in understanding the impact of interacting climate change-related abiotic factors (especially increased temperature, elevated CO2 and extremes in water availability) on the relative risks of mycotoxin contamination and impacts on food safety and security. We have thus examined the available information from the last decade on relative risks of mycotoxin contamination under future climate change scenarios and identified the gaps in knowledge. This has included the available scientific information on the ecology, genomics, distribution of toxigenic fungi and intervention strategies for mycotoxin control worldwide. In addition, some suggestions for prediction and prevention of mycotoxin risks are summarized together with future perspectives and research needs for a better understanding of the impacts of climate change scenarios.

Grafting is routinely implemented in modern agriculture to manage soilborne pathogens such as fungi, oomycetes, bacteria, and viruses of solanaceous crops in a sustainable and environmentally friendly approach. Some rootstock/scion combinations use specific genetic resistance mechanisms to impact also some foliar and airborne pathogens, including arthropod or contact-transmitted viruses. These approaches resulted in poor efficiency in the management of plant viruses with superior virulence such as the strains of tomato spotted wilt virus breaking the Sw5 resistance, strains of cucumber mosaic virus carrying necrogenic satellite RNAs, and necrogenic strains of potato virus Y. Three different studies from our lab documented that suitable levels of resistance/tolerance can be obtained by grafting commercial tomato varieties onto the tomato ecotype Manduria (Ma) rescued in the framework of an Apulian (southern Italy) regional program on biodiversity. Here we review the main approaches, methods, and results of the three case studies and propose some mechanisms leading to the tolerance/resistance observed in susceptible tomato varieties grafted onto Ma as well as in self-grafted plants. The proposed mechanisms include virus movement in plants, RNA interference, genes involved in graft wound response, resilience, and tolerance to virus infection.

Anaerobic digestion represents an interesting approach to produce biogas from organic waste materials contaminated by mycotoxins. In this study a shotgun metagenomic analysis of lab-scale bioreactors fed with mycotoxin-contaminated silage has been carried out to characterize the evolution of microbial community under the operating conditions and the key enzymatic activities responsible for mycotoxin degradation. The study was conducted at two different level of contamination for fumonisins and aflatoxin B1. After 15 days biogas production was not influenced by the presence of mycotoxins. Metagenomic analysis revealed that a high contamination rate of mycotoxins interfere with microbial diversity. Degradation of mycotoxins accounted in about 54% for aflatoxin B1 and 60% for fumonisins. The degradation activity of fumonisins resulted in the presence of partially hydrolyzed forms in both tested contamination levels. Accordingly, metagenomic functional analysis revealed the presence of two new carboxylesterase genes belonging to D. bacterium and P. bacterium putatively involved in fumonisin degradation.

Fumonisins contamination of food commodities is a worldwide problem, especially for maize. The ability to produce fumonisinsis a trait of several species of Fusarium, mainly F. verticillioides and F. proliferatum on maize, and some Aspergillus species.A. nigerand its sister speciesA. welwitschiae, can contribute to fumonisin B2(FB2) accumulation in maize kernels, although to a lesser extent than fumonisin-producingFusariumspecies. Fumonisins risk monitoring represents an effective strategy in the integrated approach for mycotoxin risk management and reduction. The availability of a user-friendlymolecular assay for the detection oftoxigenic fungal species represents a valuable tool in understanding and managing upcoming mycotoxin contamination. In this study, we developed a LAMP assay, based on the detection offum10, for a rapid and specific molecular detection of FB2-producingA. nigerandA. welwistchiae, potentially useful to perform monitoring directly “on site” in maize chain. Results showed that very low amounts of conidia are suitable to detect the presence of the target gene, thus providing information about the presence of FB2-producingAspergillusspecies and the possible upcoming fumonisins contamination in maize. The assay was combined with a suitable protocol for “in field”crude DNA extraction and a colorimetric method for easy naked-eye evaluationof results, offering a reliable and user-friendly tool to support effective reduction strategies of mycotoxin contamination in crop management programs.

Grafting of commercial tomato varieties and hybrids on the tomato ecotype Manduria resulted in high levels of tolerance to the infection of Sw5 resistance-breaking strains of tomato spotted wilt virus and of severe cucumber mosaic virus strains supporting hypervirulent satellite RNAs that co-determine stunting and necrotic phenotypes in tomato. To decipher the basis of such tolerance, here we used a RNAseq analysis to study the transcriptome profiles of the Manduria ecotype and of the susceptible variety UC82, and of their graft combinations, exposed or not to infection of the potato virus Y recombinant strain PVYC-to. The analysis identified graft- and virus-responsive mRNAs differentially expressed in UC82 and Manduria, which led to an overall suitable level of tolerance to viral infection confirmed by the appearance of a recovery phenotype in Manduria and in all graft combinations. The transcriptome analysis suggested that graft wounding and viral infection had diverging effects on tomato transcriptome and that the Manduria ecotype was less responsive than the UC82 to both graft wounding and potyviral infection. We propose that the differential response to the two types of stress could account for the tolerance to viral infection observed in the Manduria ecotype as well as in the susceptible tomato variety UC82 self-grafted or grafted on the Manduria ecotype.

Toxigenic fungi and mycotoxins are very common in food crops, with noticeable differences in their host specificity in terms of pathogenicity and toxin contamination. In addition, such crops may be infected with mixtures of mycotoxigenic fungi, resulting in multi-mycotoxin contamination. Climate represents the key factor in driving the fungal community structure and mycotoxin contamination levels pre- and post-harvest. Thus, there is significant interest in understanding the impact of interacting climate change-related abiotic factors (especially increased temperature, elevated CO2 and extremes in water availability) on the relative risks of mycotoxin contamination and impacts on food safety and security. We have thus examined the available information from the last decade on relative risks of mycotoxin contamination under future climate change scenarios and identified the gaps in knowledge. This has included the available scientific information on the ecology, genomics, distribution of toxigenic fungi and intervention strategies for mycotoxin control worldwide. In addition, some suggestions for prediction and prevention of mycotoxin risks are summarized together with future perspectives and research needs for a better understanding of the impacts of climate change scenarios.

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.

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.

Fusarium proliferatum causes diverse diseases of many economically important plants. The fungus produces several mycotoxins of which the fumonisins are the most toxic. Currently, deletion of key genes for mycotoxin biosynthesis is a laborious and time-consuming procedure. We developed a novel CRISPR/Cas9-based genome-editing tool for the direct delivery of preassembled Cas9 ribonucleoproteins into protoplasts of F. proliferatum. Our CRISPR–Cas9 system couples a site-specific double-strand DNA break mediated by two Cas9 ribonucleoproteins with microhomology recombination requiring only 50-bp regions flanking the target gene. This system reduces the risk of off-target mutations and minimizes the risk of altering any gene adjacent to the target region. We used this tool to delete a polyketide synthase gene (FUM1) required for fumonisin biosynthesis. The mutants generated are no longer able to produce fumonisins, confirming the key role of FUM1 in fumonisin biosynthesis. Our CRISPR-Cas9 system is an important new tool for genetic studies of Fusarium.

Little is known on the impact that climate change (CC) may have on Aspergillus carbonarius and Ochratoxin A (OTA) contamination of grapes, especially in the Mediterranean region where in CC scenarios temperature are expected to increase by +2-5 °C and CO

A transcriptome analysis was produced from tomato roots inoculated with the hyphomycete Pochonia chlamydosporia at three different times. Gene expression data were also yielded from fungus grown in vitro or endophytic. A next-generation sequencing (NGS) and network analysis approach were applied. We identified 3.676 differentially expressed tomato genes (DEG), highlighting a core of 93 transcripts commonly down- or upregulated at every time point, shedding light on endophytism process. Functional categories related to plant information-processing system, which recognizes, percepts, and transmits signals, were associated with gene upregulated early in time, with higher representations in processes such as plant defense regulation later in time. Network analysis of a DEG subset showed dominance of MAP kinase hubs in the uninoculated control samples, replaced by an increased centrality of WRKY transcription factor and ETR-ethylene response factor genes in the colonized roots. Fungus genes expressed during progression of plant colonization, therefore related to the host colonization process or endophytism persistence, were also identified. Data provided a high-resolution insight on tomato transcriptome changes as induced by endophytism, highlighting a specific modulation of stress-responsive transcripts, related to a selective activation of defense pathways, likely required by the fungus to establish a persistent endophytic lifestyle.

Ochratoxin A (OTA) is a potent pentaketide nephrotoxin widely contaminating food and feed products (grains, legumes, coffee, dried fruits, meat derived products, beer and wine). OTA is the primary mycotoxin risk in wine and dried vine fruits. The contamination of grapes and wine is strongly related to climatic conditions during cultivation. There is little information on the impact that climate change environmental fluxes may have on fungal colonization and ochratoxin A contamination of grapes. It has been suggested that the Mediterranean region is a hot spot for the impact of climate change scenarios with temperatures expected to increase by +2-5°C and CO2 to double or triple (400 ppm vs 800/1200 ppm) in the medium term. The northern region of Apulia, in the south of Italy, is normally considered to be an area where the risk of OTA in vineyards is relatively low. This study has examined the effect of i) increased temperature in the alternating day/night cycle (15-28°C vs 18-34°C), ii) existing and predicted CO2 concentrations (400 vs 1000 ppm), iii) conditions where water is freely available or drought stress is imposed (0.99 vs 0.93 aw ) in A. carbonarius strains (ITEM 7444, ITEM 5010 and B7), grown on grape-based matrices, by analysis of growth rate, OTA production and expression of key toxin biosynthetic genes. The results showed that in both non-stressed aw and drought stressed conditions the effect of elevated CO2 (1000 ppm) caused a general positive stimulation of growth rates at both the temperature cycles. Furthermore, while differences in the alternating day/night temperatures at 400 ppm and 0.99 aw did not result in any variation in OTA production, elevated CO2 condition (1000 ppm) was observed to stimulate OTA production especially at the 15-28°C cycle. These results were confirmed by gene expression analysis showing higher expression levels of OTA related genes at higher CO2 concentration. This study showed for the first time that elevated CO2 concentrations under different fluxes in day/night temperatures in the Mediterranean region may result in an increasing of OTA risk contamination in the production chain, being grapes destined for wine production less resilient under climate change scenarios.

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

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

Fungal starter, such as Penicillium nalgiovense , are commonly used to inoculate sausages before seasoning process. However, Penicillium 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.

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

In this study we describe the genetic elements and the antimicrobial resistance units (RUs) harboured by the Salmonella Typhimurium monophasic variant 1,4,[5],12:i:- strain ST1030. Of the three identified RUs two were chromosomal, RU1 (IS26-blaTEM-1-IS26-strAB-sul2- IS26) and RU2 (IS26-tetR(B)-tetA(B)-ΔIS26), and one, RU3 (a sul3-associated class 1 integron with cassette array dfrA12-orfF-aadA2-cmlA1-aadA1), was embedded in a Tn21-derived element harboured by the conjugative I1 plasmid pST1030-1A. IS26 elements mediated the antimicrobial resistance gene (ARG) shuffling and this gave rise to pST1030-1A derivatives with different sets of ARGs. ST1030 also harboured two ColE1-like plasmids of which one, pST1030-2A, was mobilisable and the target of an intracellular translocation of the Tn21-derived element; the second (pST1030-3) was an orphan mob-associated oriT plasmid co-transferred with pST1030-1A and pST1030-2A. pST1030-2A and pST1030-3 also carried a parA gene and a type III restriction modification system, respectively. Overall analysis of our data reinforces the role played by IS26, Tn21-derived elements and non-conjugative plasmids in the spread of ARGs and supplies the first evidence, at least in Salmonella, for the identification of a natural isolate harbouring a three-element mobilisation system in the same cell.

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

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 in vitro 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 in vitro resistance possessed a mutation at the Sdh B gene, whereas all isolates resistant to carbendazim and iprodione possessed mutations at β-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.

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. References 1. 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.

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 introduces 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.

Polyketide synthase (PKSs) and nonribosomal peptide synthetase (NRPSs) are large multimodular enzymes involved in biosynthesis of polyketide and peptide toxins produced by fungi. Furthermore, hybrid enzymes, in which a reducing PKS region is fused to a single NRPS module, are also responsible of the synthesis of peptide-polyketide metabolites in fungi. The genes encoding for PKSs and NRPSs have been exposed to complex evolutionary mechanisms, which have determined the great number and diversity of metabolites. In this study, we considered the most important polyketide and peptide mycotoxins and, for the first time, a phylogenetic analysis of both PKSs and NRPSs involved in their biosynthesis was assessed using two domains for each enzyme: ?-ketosynthase (KS) and acyl-transferase (AT) for PKSs; adenylation (A) and condensation (C) for NRPSs. The analysis of both KS and AT domains confirmed the differentiation of the three classes of highly, partially and non-reducing PKSs. Hybrid PKS-NRPSs involved in mycotoxins biosynthesis grouped together in the phylogenetic trees of all the domains analyzed. For most mycotoxins, the corresponding biosynthetic enzymes from distinct fungal species grouped together, except for PKS and NRPS involved in ochratoxin A biosynthesis, for which an unlike process of evolution could be hypothesized in different species.