Laccases (LCs) are multicopper oxidases that find application as versatile biocatalystsfor the green bioremediation of environmental pollutants and xenobiotics. In this study weelucidate the degrading activity of Lac2 pure enzyme form Pleurotus pulmonarius towards aflatoxinB1 (AFB1) and M1 (AFM1). LC enzyme was purified using three chromatographic steps andidentified as Lac2 through zymogram and LC-MS/MS. The degradation assays were performedin vitro at 25 °C for 72 h in buffer solution. AFB1 degradation by Lac2 direct oxidationwas 23%. Toxin degradation was also investigated in the presence of three redox mediators,(2,20-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) (ABTS) and two naturally-occurring phenols,acetosyringone (AS) and syringaldehyde (SA). The direct effect of the enzyme and the mediatedaction of Lac2 with redox mediators univocally proved the correlation between Lac2 activity andaflatoxins degradation. The degradation of AFB1 was enhanced by the addition of all mediators at10 mM, with AS being the most effective (90% of degradation). AFM1 was completely degraded byLac2 with all mediators at 10 mM. The novelty of this study relies on the identification of a pureenzyme as capable of degrading AFB1 and, for the first time, AFM1, and on the evidence that themechanism of an effective degradation occurs via the mediation of natural phenolic compounds.These results opened new perspective for Lac2 application in the food and feed supply chains as abiotransforming agent of AFB1 and AFM1

Milk protein crosslink through the action of enzymes represents a feasible strategy to impart new functionalities to cheese. In this work we reported the effects of a laccase mediator system (LMS) on protein crosslink and antioxidant property of curd. The crosslinking activity of a purified recombinant laccase Ery4 and a commercial enzyme preparation (cLC), with three mediators was firstly evaluated in milk and then applied before curd manufacture. Only Ery4-LMS significantly increased curd weight compared to that of the control sample. SDS-PAGE revealed that similar high molecular weight bands produced by both LMSs in milk were also retained in curds. The antioxidant activity recorded in curds with Ery4-LMS was the highest among all samples both before and after gastro-pancreatic digestion. This is the first time that a CGA-based LMS is used in manufacture of curd with improved antioxidant properties. These results open new perspectives for dairy applications.

La laccasi (LC) è un enzima ampiamente studiato per modulare le proprietà reologiche e tecnologiche degli alimenti. L'applicazione in latte e prodotti lattiero caseari è tuttavia limitata. Recentemente è stato studiato il suo ruolo nella degradazione in latte dell'aflatossina M1, oltre che nella creazione di crosslink proteici per migliorare la texture e la reologia dei prodotti lattiero caseari. Quest' ultima sua proprietà potrebbe essere sfruttata per favorire il recupero di proteine dal siero durante la caseificazione aumentando la resa casearia. D'altra parte l'applicazione della LC richiede la combinazione con l'acido clorogenico (CGA), un polifenolo naturale che potrebbe conferire al formaggio prodotto nuove caratteristiche qualitative, come un'aumentata attività antiossidante.Pertanto, l'obiettivo di questo lavoro è stato valutare l'aumento in resa casearia e attività antiossidante della cagliata ottenuta pretratrattando il latte per 45' a 38°C con concentrazioni note di LC + CGA prima dell'utilizzo del caglio. La cagliata ottenuta è stata comparata a quella controllo non trattata valutando anche il profilo elettroforetico su SDS-PAGE, e l'attività antiossidante dopo digestione gastrico-pancreatica simulata.Il trattamento del latte con il sistema laccasi-acido clorogenico ha causato la formazione di oligomeri ad alto e medio peso molecolare (>200 e 50 kDa, rispettivamente) rilevabili in SDS-PAGE. Parallelamente, le bande associate alle ?-, ?- e ?-caseine, hanno subito una lieve ma significativa riduzione di intensità, suggerendo un loro coinvolgimento nella formazione dei suddetti oligomeri, confermato successivamente anche dall'analisi in cromatografia liquida associata a spettrometria di massa. Questo pattern elettroforetico, osservato in latte prima dell'aggiunta del caglio, è stato osservato invariato anche nelle cagliate prodotte, indice che tali oligomeri sono incorporati nella rete caseinica al momento della coagulazione. Contrariamente della transglutaminasi, che modifica il sito di taglio della ?-caseina, le modalità d'impiego del sistema LC + CGA non hanno invece influenzato l'azione del caglio, suggerendo pertanto una sua facile implementazione nel processo di caseificazione industriale. La cagliata ottenute hanno inoltre mostrato una resa casearia significativamente superiore, circa 25%, a quella della cagliata controllo sia in peso fresco (2.89g vs 2.14g) che in peso secco (1.66g vs 1.28g), presumibilmente ascrivibile all'aumento totali del contenuto proteico totale.La cagliata trattata con il sistema LC + CGA, sottoposta a digestione gastrico pancreatica simulata, ha mostrato un aumento dell'attività antiossidante di quasi 4 volte rispetto a quella del controllo non trattato (2273.3 vs 586 µmol TEAC/L). I dati presentati dimostrano come un unico trattamento enzimatico, facilmente implementabile nel processo produttivo, potrebbe apportare un significativo contributo in campo lattiero caseario su più fronti. L'azione degrad

Fumonisins (FBs), which are carcinogenic mycotoxins, are known to be typically produced by several phytopathogenicfungal species belonging to the genus Fusarium. F. proliferatum and F. verticillioides, two important pathogens of maizeworldwide, are the most common species that produce FBs. The main FBs produced by these species are FB1, FB2 and FB3.Moreover, recently, fungal strains belonging to Aspergillus niger have been also reported to produce FBs (in particular, FB2and FB4). In a survey on maize carried out in Central Italy, 17 maize kernel samples were collected at harvest and analysedfor FB1, FB2 and FB3, as well as fungal contamination, with a particular attention to the species-producing FBs. All 17samples were contaminated by F. verticillioides and/or F. proliferatum at a level ranging from 13% to 100% of kernels.However, 10 out of 17 samples were also contaminated by Aspergillus section Nigri with a range from 6% to 68% ofkernels. There was a significant inverse logarithmic relationship between levels of Fusarium and Aspergillus contamination.All samples were contaminated by FBs; FB1 ranged from 0.09 to 30.2 ?g g-1, whereas FB2 ranged from 0.04 to 13.2 ?g g-1.The ratio of FB2/FB1 contamination in the maize samples was evaluated and the highest values occurred in samplescontaminated with Aspergillus section Nigri. Thirty strains of Aspergillus section Nigri isolated from these samples weremolecularly identified (based on sequences of two housekeeping genes) and analysed for their capability to produce FB2.Among the 30 strains isolated, 12 were identified as Aspergillus welwitschiae (syn. A. awamori) and 18 as A. tubingensis.FB2 was produced by five out of 12 strains of A. welwitschiae within a range of 0.20-5 ?g g-1. This is the first reportshowing the capability of Aspergillus section Nigri from maize to produce FB2 and its possibility to contribute to FBaccumulation in kernels.

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).

Ochratoxin A (OTA) is a mycotoxin with a main nephrotoxic activity contaminatingseveral foodstuffs. In the present report, five soil samples collected from OTA-contaminatedvineyards were screened to isolate microorganisms able to biodegrade OTA. When cultivated inOTA-supplemented medium, OTA was converted in OTalpha by 225 bacterial isolates. To revealclonal relationships between isolates, molecular typing by using an automated rep-PCR systemwas carried out, thus showing the presence of 27 different strains (rep-PCR profiles). The16S-rRNA gene sequence analysis of an isolate representative of each rep-PCR profiles indicatedthat they belonged to five bacterial genera, namely Pseudomonas, Leclercia, Pantoea, Enterobacter, andAcinetobacter. However, further evaluation of OTA-degrading activity by the 27 strains revealedthat only Acinetobacter calcoaceticus strain 396.1 and Acinetobacter sp. strain neg1, consistentlyconserved the above property; their further characterization showed that they were able to convert82% and 91% OTA into OTalpha in six days at 24 °C, respectively. The presence of OTalpha, asthe unique OTA-degradation product was confirmed by LC-HRMS. This is the first report onOTA biodegradation by bacterial strains isolated from agricultural soils and carried out underaerobic conditions and moderate temperatures. These microorganisms might be used to detoxifyOTA-contaminated feed and could be a new source of gene(s) for the development of a novelenzymatic detoxification system.

During their life cycle, plants can undergo simultaneous attack by different pathogens that produce various toxins. It is well known that in some plant-fungal interactions, mycotoxins play an important role in pathogenesis and induce a reactive oxygen species increase. Plants counteract the overaccumulation of reactive oxygen species by reinforcing their defence systems. The mycotoxins T-2 toxin (T-2) and beauvericin (BEA) are produced by some Fusarium species and have different chemical structures, mechanisms of action and biological activities. In this study, the individual and combined effects of these two toxins on defence systems, such as the ascorbate-glutathione cycle and peroxidases, were evaluated in cherry tomato shoots. Hydrogen peroxide content as an index of oxidative stress was also measured. Inhibitory effects on ascorbate peroxidase, dehydroascorbate reductase and ascorbate, and stimulatory effects on glutathione reductase, monodehydroascorbate reductase and reduced glutathione were observed when tomato plants were simultaneously treated with BEA and T-2. The trend of these biochemical parameters highlight the presence of a range of defence mechanisms activated by plants in response to mycotoxins. The interaction between BEA and T-2 resulting in synergistic and/or antagonistic effects on the studied defence systems is also discussed. It is concluded that the effects of these mycotoxins alone are not predictive of their combined effects.

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

Currently, there is very little information available regarding the microbiome associated with the wine production chain. Here, we used an amplicon sequencing approach based on high-throughput sequencing (HTS) to obtain a comprehensive assessment of the bacterial community associated with the production of three Apulian red wines, from grape to final product. The relationships among grape variety, the microbial community, and fermentation was investigated. Moreover, the winery microbiota was evaluated compared to the autochthonous species in vineyards that persist until the end of the winemaking process. The analysis highlighted the remarkable dynamics within the microbial communities during fermentation. A common microbial core shared among the examined wine varieties was observed, and the unique taxonomic signature of each wine appellation was revealed. New species belonging to the genus Halomonas were also reported. This study demonstrates the potential of this metagenomic approach, supported by optimized protocols, for identifying the biodiversity of the wine supply chain. The developed experimental pipeline offers new prospects for other research fields in which a comprehensive view of microbial community complexity and dynamics is desirable.

Ochratoxin A (OTA) is a mycotoxin denoted by a nephrotoxic activity contaminating several foodstuffs. Nowadays, the biological systems for OTA degradation to the less toxic OT? aroused great interest by the scientific community. In the present study, bacteria able to biodegrade OTA were isolated from soil samples collected in OTA-contaminated vineyards. Soil samples were collected from five vineyards of Negroamaro and Primitivo grape cultivars in Salento (Southern Italy). They were cultured in appropriate media added with OTA, mycotoxin degradation was determined by HPLC/FLC analysis and bacterial colonies were isolated by plating. Clonal relationships between isolates was assessed by using an automated rep-PCR system and then each strain was identified by 16S rRNA gene sequencing. A total of 225 bacterial isolates were able to convert OTA in OT?. The molecular analysis of the above isolates showed the presence of 27 different strains (rep-PCR profiles). The sequence analysis of the 16S-rRNA gene indicated that they belonged to five bacterial genera: Pseudomonas, Leclercia, Pantoea, Enterobacter and Acinetobacter. Additional assessment of OTA-degrading capacity of the 27 strains indicated that only the Acinetobacter calcoaceticus strain 396.1 and the A. sp. strain neg1 conserved the above property: both strains were further studied thus showing that they were able to convert 82% and 91% OTA into OT? in 6 days at 24°C, respectively. The occurrence of OT?, as the sole OTA-degradation product was established by LC-MS/MS.This is the first description on OTA biodegradation under aerobic conditions and moderate temperature by bacterial strains from agricultural soils. These microorganisms might be used to detoxify OTA contaminated feed and could be a resource for the development of a new enzymatic detoxification system.

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

Ochratoxin A (OTA) is a nephrotoxic and potentially carcinogenic mycotoxin produced by several species of Aspergillus and Penicillium. It is one of the major mycotoxins contaminating grain, grapes and a variety of food products, and the development of methods for reducing pre-and post-harvest contamination has drawn considerable attention. In the current study, we isolated and sequenced the genome of a novel free-living Acinetobacter strain able to degrade OTA. Biochemical studies suggest that the degradation reaction proceeds via peptide bond hydrolysis.

Pseudomonas fluorescens is a genetically and phenotypically heterogeneous species that is often reported as a spoiler of fresh foods, but it has recently been implicated in clinical infection. In this study, we sequenced the genome of P. fluorescens strain ITEM 17298, isolated from mozzarella cheese and able to cause several alterations under cold storage.

Broccoli (Brassica oleracea L. var. italica) is largely cultivated in southern Italy. It is an important source of phytonutrients, which are partially lost during postharvest storage. The aim of this work was to evaluate the overall effect of five different low-intensity light-emitting diodes (LEDs) on the quality parameters of broccoli florets over 20 d of cold storage. The level of ascorbic acid, chlorophylls, carotenoids, phenolic compounds and soluble proteins, as well as colour analysis, were evaluated. Green LED increased the chlorophyll and ascorbic acid content; white, red and yellow LEDs had a positive effect on the redox status of broccoli. Globally, only green LED had a statistically significant positive effect when considering all analysed parameters and could be proposed to prolong the shelf life of broccoli during cold storage.

At the three-leaf stage, sterile seedlings of resistant (CO433) and susceptible (CO389) maize lines was spiked with fumonisin B1 (FB1) (dissolved in PBS, pH =7.4, at the final concentration of 1mg/mL) in the part of the stem between the collar and the insertion of the first leaf. At various times, the FB1 content was determined by the use of HPLC/FLD previously derivatized with an o-phthaldeyde (OPA). The stem was ground with liquid nitrogen and extracted with a solution methanol/water (70:30, v/v). Recovery was 106% (RSD < 8%). The concentration of FB1 after 3 hours (88%, RSD 12%) and 48 hours (92%, RSD 7%) after spiking showed that no translocation in seedling maize occurred. To evaluate if the defence systems were alerted, the leaves were used to monitor the ascorbate-glutathione cycle, phenolics and enzymes protective from oxidative stress (catalase, superoxide dismutase and cytosolic peroxidases) at 3 and 48 hours post-spiking. The study was also extended to the analysis of total antioxidants, hydrogen peroxide and malondialdehyde contents to evaluate the oxidation level after FB1 treatment. Defense response promptly appeared activated in leaves of resistant line; particularly, after 3h, ascorbate, ascorbate peroxidase, SOD were augmented, underlining a higher fitness in the counteracting the phytotoxic action of FB1. In contrast, in the susceptible line, catalase, phenolics and ascorbate increased at longer time, conferring a lower readiness to the FB1 treatment. Same trend in total antioxidants, cytosolic peroxidases and other components analyzed was observed in both CO433 and CO389 after spiking. FB1, although did not cause seedlings suffering, induced metabolic perturbations. These data are useful for further investigation on molecular mechanisms that are to the basis of the FB1 and others mycotoxins contamination in maize, in order to improve resistance to fungal pathogens.

The fungus Fusarium verticillioides is a pathogen of maize and can produce the mycotoxins fumonisins. The present work examined the effect of abiotic factors on the expression of two fumonisin biosynthetic genes (FUM2 and FUM21) and fumonisin production in the fungus. Specifically, the effects of water activity (0.901-0.990), temperature (20-30°C) and incubation time (7-21 days) were analysed in cultures of F. verticillioides strains ITEM 10027 and ITEM 1744. Fumonisin B were measured through liquid chromatography tandem mass spectrometry and gene expression was quantified with relative real-time PCR, using the Livak method. Fumonisin production increased with incubation time up to 21 days and the transcription of both genes was highest at 14 days; however intraspecific variability was observed. FUM2 and FUM21 expression was positively correlated to fumonisin synthesis (p<=0.01 and 0.05) at different water activity and temperature regimes. . A sensibly higher transcription of FUM2 in comparison with FUM21 was observed for all the conditions considered. Incubation time played a significant role on fumonisin production and gene expression, with the highest contamination and the highest gene expression respectively after 21 and 14 days of incubation. Temperature significantly affected only FUM21 expression, with optimum at 25°C; while water activity did not have a significant effect.

Members of the fungal genus Fusarium can produce numerous secondary metabolites, including the nonribosomal mycotoxins beauvericin (BEA) and enniatins (ENNs). Both mycotoxins are synthesized by the multifunctional enzyme enniatin synthetase (ESYN1) that contains both peptide synthetase and S-adenosyl-l-methionine-dependent N-methyltransferase activities. Several Fusarium species can produce ENNs, BEA or both, but the mechanism(s) enabling these differential metabolic profiles is unknown. In this study, we analyzed the primary structure of ESYN1 by sequencing esyn1 transcripts from different Fusarium species. We measured ENNs and BEA production by ultra-performance liquid chromatography coupled with photodiode array and Acquity QDa mass detector (UPLC-PDA-QDa) analyses. We predicted protein structures, compared the predictions by multivariate analysis methods and found a striking correlation between BEA/ENN-producing profiles and ESYN1 three-dimensional structures. Structural differences in the ? strand's Asn789-Ala793 and His797-Asp802 portions of the amino acid adenylation domain can be used to distinguish BEA/ENN-producing Fusarium isolates from those that produce only ENN.

DNA phylogenetic comparisons have shown that morphology-based species recognition often underestimates fungal diversity. Therefore, the need for accurate DNA sequence data, tied to both correct taxonomic names and clearly annotated specimen data, has never been greater. Furthermore, the growing number of molecular ecology and microbiome projects using high-throughput sequencing require fast and effective methods for en masse species assignments. In this article, we focus on selecting and re-annotating a set of marker reference sequences that represent each currently accepted order of Fungi. The particular focus is on sequences from the internal transcribed spacer region in the nuclear ribosomal cistron, derived from type specimens and/or ex-type cultures. Re-annotated and verified sequences were deposited in a curated public database at the National Center for Biotechnology Information (NCBI), namely the RefSeq Targeted Loci (RTL) database, and will be visible during routine sequence similarity searches with NR_prefixed accession numbers. A set of standards and protocols is proposed to improve the data quality of new sequences, and we suggest how type and other reference sequences can be used to improve identification of Fungi.

Fumonisins are mycotoxins with cancerpromotingactivity and are associated with a number ofanimal and human diseases. The potential risk of contaminationby fumonisin B2 (FB2), although at low levels, hasbeen demonstrated in must and wine. Black aspergilli ingeneral and Aspergillus niger in particular are considered tobe the major responsible agents of FB2 contamination ingrape and its by-products. Contamination by FB2 thereforeis yet another safety concern of grape and wine producers,as ochratoxin A, produced mainly by A. carbonarius, mayprove to be a major mycotoxicological problem in thegrape-wine chain.

Comparisons of draft genome sequences of three geographically distinct isolates of Fusariumfujikuroi with two recently published genome sequences from the same species suggest diverseprofiles of secondary metabolite production within F. fujikuroi. Species- and lineage-specific genes,many of which appear to exhibit expression profiles that are consistent with roles in host-pathogeninteractions and adaptation to environmental changes, are concentrated in sub-telomeric regions.These genomic compartments also exhibit distinct gene densities and compositional characteristicswith respect to other genomic partitions, and likely play a role in the generation of moleculardiversity. Our data provide additional evidence that gene duplication, divergence and differentialloss play important roles in F. fujikuroi genome evolution and suggest that hundreds of lineagespecific genes might have been acquired through horizontal gene transfer.

Trichoderma atrobrunneum F.B. Rocha, P. Chaverri& W. Jaklitsch strain ITEM 908 (formerly known as T. harzianum ITEM 908), is a biocontrol strain that is being registered under the European Union regulation as an active ingredient for the production of commercial biopesticides. The strain ITEM 908 proved to be able to inhibit completely the formation of perithecia by Fusarium graminearum in dual cultures and to release in the agar medium metabolites that reduce the number of perithecia by over 70% (Altomare et al., poster presentation in this Congress). Therefore, ITEM 908 appears to be a good candidate biocontrol strain for prevention of Fusarium Head Blight (FHB) in the field by treatment of plant residues of the preceeding crop, thus reducing the primary inoculum. For the univocal characterization of this commercially valuable isolate and as a base for further studies aimed at elucidating its mechanisms of action and its physiological and molecular interactions with plants and target pathogens and pests, we sequenced the whole genome of the strain ITEM 908. The genome was sequenced on an Ion S5 platform generating around 7M bpand assembled using the Spades v5.0 software. The resulting genome sequence has an estimated size of 39,131,654 bp. The reference gene sequences of ITS and TEF1 were extracted from the genome assembly. The identification of the strain ITEM 908 at species level was obtained by phylogenetic analysis with Maximum likelihood (ML) analysis performed with one-hundred ITS-TEF1 manually concatenated dataset retrieved from sequences of Trichoderma spp. deposited in GenBank. The analysis placed ITEM 908 within the T. atrobrunneum group, close to T. afroaharzianum and T. guizhouense. Genome was annotated using the Augustus v3.1 software and 8649 genes were predicted. Approximately 3000 different pfam domains were detected and used to group genes within functional categories, including glycoside hydrolases, proteases and gene involved in stress tolerance and in secondary metabolites biosynthesis. Among these, 20 putative PKS, 8 putative NRPS and 5 putative PKS-NRPS were identified. Moreover, the secretome of T. atrobrunneum ITEM 908, consisting of 761 proteins, was in silico predicted by the software SignalP (http://www.cbs.dtu.dk/services/SignalP/), that detects the presence of the secretion signal peptide at the N-terminus in amino acid sequences of a protein.The preliminary analysis of predicted genes highlights the potential ability of T. atrobrunneum ITEM 908 to produce a broad range of enzymes involved in the biocontrol activity

Advanced age is characterized by several changes, one of which is the impairment of the homeostasis of intestinal microbiota. These alterations critically influence host health and have been associated with morbidity and mortality in older adults. "Inflammaging," an age-related chronic inflammatory process, is a common trait of several conditions, including sarcopenia. Interestingly, imbalanced intestinal microbial community has been suggested to contribute to inflammaging. Changes in gut microbiota accompanying sarcopenia may be attenuated by supplementation with pre- and probiotics. Although muscle aging has been increasingly recognized as a biomarker of aging, the pathophysiology of sarcopenia is to date only partially appreciated. Due to its development in the context of the age-related inflammatory milieu, several studies favor the hypothesis of a tight connection between sarcopenia and inflammaging. However, conclusive evidence describing the signaling pathways involved has not yet been produced. Here, we review the current knowledge of the changes in intestinal microbiota that occur in advanced age with a special emphasis on findings supporting the idea of a modulation of muscle physiology through alterations in gut microbial composition and activity.

In a survey carried out on 87 rotted fig fruits samples collected in the Apulia region of Italy, the authors isolated 126 Fusarium strains identified as F. ramigenum (69 strains), F. solani (49), F. proliferatum (five) and three not identified. Investigation on the fertility of the strains belonging to F. proliferatum and F. ramigenum revealed that only strains of F. proliferatum were fertile. The identity of F. ramigenum strains was confirmed by sequencing a portion of the translation elongation factor-1 gene. When Fusarium species were analysed for their toxigenicity, 37/69 strains of F. ramigenum produced fusaric acid (FA) up to 525 mg kg-1; 30 strains produced beauvericin (BEA) up to 190 mg kg-1; 60 strains produced fumonisin B1 (FB1) and fumonisin B2 (FB2) up to 1575 mg kg-1 of total FBs; and two strains produced fusaproliferin (FUP) up to 345 mg kg-1; all five strains of F. proliferatum produced FA at low levels; two strains produced BEA up to 205 mg kg-1; one strain produced FB1 and FB2, 1100 and 470 mg kg-1, respectively; and one strain produced FUP, 820 mg kg-1; F. solani (30 strains) produced FA, 13 strains up to 215 mg kg-1. Few fungal extracts showed high toxicity toward brine shrimp larvae and in some cases in relation to BEA and FA content. A pathogenic assay on fig fruits showed that all three species were pathogenic, with higher virulence of F. ramigenum. These data report for the first time the production of BEA and FB1/FB2 by F. ramigenum and show that it is a main agent of fig endosepsis in Apulia and can contribute to fumonisin contamination of fresh and dried figs.

Mycotoxin contamination of staple food commodities is a relevant health and economic issue worldwide.The development of green and effective reduction strategies to counteract the contamination by multiplemycotoxins has become an urgent need. The aim of this work was to evaluate the capability of a laccase(LC) from Pleurotus eryngii and a laccase-mediator systems (LMSs) to degrade aflatoxin B1 (AFB1),fumonisin B1 (FB1), ochratoxin A (OTA), deoxynivalenol (DON), Zearalenone (ZEN) and T-2 toxin in in vitroassays. In addition, the simultaneous mycotoxin degradation capability with selected LMSs was evaluatedwith combinations of AFB1 and ZEN, and FB1 and T-2 toxin. Redox mediators were found to drasticallyincreasethe degradation efficiencies of the enzyme. AFB1, FB1, OTA, ZEN and T-2 toxin degradation by thebest performing LMS were 73%, 74%, 27%, 100% and 40%, respectively. No degradation was registered forDON. Notably, AFB1 and ZEN were simultaneously degraded by 86% and 100%, while FB1 and T-2 by 25%and 100%, respectively. LMS proved to be a promising approach to enhance degradation properties of LCenzymes and for the potential development of a multi-mycotoxin reducing method.

Powdery mildew (PM), caused by the fungus Erysiphe necator, is one of the most widespread fungal disease of grape and maycause extensive openings on the berry surface during the infection. We evaluated the effect of damage caused by PM in grape berrieson the growth of and mycotoxin production by Aspergillus and on the oxidative stress in infected berries. Berries of Vitis vinifera L.cv. Negroamaro with sound skin (SS) and those naturally infected by PM were surface sterilized and inoculated with eitherfumonisin B2 (FB2)-producing strains of Aspergillus niger or ochratoxin A (OTA)-producing strains of Aspergillus carbonariusand incubated at 20 and 30uC. The PM berries were significantly more susceptible to both Aspergillus colonization (5 to 15 timesmore susceptible) and OTA and FB2 contamination (2 to 9 times more susceptible) than were SS berries. The highest toxinconcentration was detected in inoculated PM berries both for OTA (9 ng/g) at 20uC and for FB2 (687 ng/g) at 30uC. In inoculated SSand PM berries, although malondialdehyde and hydrogen peroxide concentrations did not increase, the two black Aspergillusspecies caused a significant decrease in ascorbate content, thus inducing a pro-oxidant effect. These results indicate that grape berriesaffected by PM are more susceptible to black Aspergillus growth and to production and/or accumulation of FB2 and OTA. Thus,preventive control of E. necator on grape berries could reduce the mycotoxicological risk from black Aspergillus infection.

Aspergillus niger is a fungus able to produce the carcinogenic mycotoxins ochratoxin A (OTA) and fumonisins. We analysed the influence of light of various wavelengths on growth, conidiation, fumonisin B2 (FB2) and OTA biosynthesis by A. niger ITEM 7097. Light from both sides of the spectrum, from long (627 nm) to short wavelengths (470-455 nm), had a stimulating effect on growth, with the highest stimulation under blue (455 nm, 1,700 Lux) and short-wave blue light (390 nm). Conidiation was reduced by 40% under a short blue wavelength (455 nm, 200 Lux), but strongly promoted under light at an even shorter wavelength (390 nm), with an increase of about 200 fold in comparison to the dark. Production of FB2 and OTA was mutually regulated by light. FB2 production was promoted under light conditions: red and blue light in particular increased FB2 biosynthesis by 40%. Conversely, OTA production was greatly inhibited under red and blue light in comparison to dark incubation, with a mean reduction of about 40 fold, indicating a reverse regulation of both biosynthetic pathways. Incubation under a 390 nm wavelength repressed the production of both toxins to non-detectable levels.

Light is a very important signal for fungi since it influences many different physiological responses. We analyzed the influence of light of varying wavelength and intensity on growth, conidiation and biosynthesis of fumonisin B(1) (FB(1)), B(2) (FB(2)), and B(3) (FB(3)) by Fusarium verticillioides ITEM 10027. Wavelengths across the visible spectrum, from red (627 nm) to blue (470-455 nm), stimulated the growth and increased the fumonisin production, by up to 150 % over dark incubation. If the intensity of the 455 nm blue light increased from 200 to 1700 lx, the fumonisin biosynthesis decreased. Incubation under a short wave blue light (390 nm) showed reduced fungal growth and fumonisin production by up to 85 %. White pulsing light had no effect on growth but reduced fumonisin production to half of what observed during dark incubation. Real time reverse transcriptase (RT)-PCR was used to measure the expression level of Fum1, Fum21 and FvVE1 transcripts, which encode proteins involved in fumonisin biosynthesis. There was a significant correlation between gene expression and fumonisin production.

Ophiobolin A (O-A) is a sesterpenoid with numerous biological activities, including potential anticancer effects. Its production at an industrial level is hampered due to inability of fungus Bipolaris maydis to biosynthesise it in vitro in large amount. Among the environmental factors regulating fungal metabolism, light plays a crucial role. In this study, the use of different light wavelength (light emitting diodes (LEDs)) was evaluated to increase the O-A production. The white light allowed the highest production of the metabolite. The blue and green lights showed an inhibitory effect, reducing the production to 50%, as well as red and yellow but at a lower level. No correlation between fungal growth and metabolite production was found in relation to the light type. A novel application of LED technologies, which can be optimised to foster specific pathways and promote the production of metabolites having scientific and industrial interest was proposed.

Fumonisins are a group of mycotoxins, mainly found in maize and maize-based food and feed, associated with several diseases in animals. The impact of these toxins on the economy and health worldwide has driven several efforts to clarify the role of environmental factors that can influence fumonisin biosynthesis by the toxigenic species. We analyzed the influence of light of varying wavelength on growth and fumonisin biosynthesis by the fungus Fusarium proliferatum ITEM 1719. Light in general had a positive influence on growth, with a mean increase of the grow rate of about 40% under light exposure in comparison to the dark incubation. Wavelengths from both sides of the spectrum, from long (627 nm) to short wavelength (470-455 nm) had a stimulating effect on fumonisin biosynthesis compared to the dark incubation: fumonisins B(1) (FB(1)) and B(2) (FB(2)) production increased of about 40 fold under red, 35 fold under blue, 20 fold under royal blue, 10 fold under green, 5 fold under yellow and 3 fold under white light in comparison to the dark incubation. The transcriptional regulation of the FUM1 fumonisin biosynthesis gene was analyzed by Real time reverse transcriptase PCR quantification, revealing a correlation between fumonisin biosynthesis and gene expression. These findings show a role of light on the growth and the modulation of fumonisin biosynthesis and provide new information on the physiology of an important toxigenic maize pathogen.

Mycotoxins are secondary metabolites produced by toxigenic fungi contaminating foods and feeds in pre-, postharvestand processing, and represent a great concern worldwide, both for the economic implications and for thehealth of the consumers. Many environmental conditions are involved in the regulation of mycotoxin biosynthesis.Among these, light represents one of the most important signals for fungi, influencing several physiologicalresponses such as pigmentation, sexual development and asexual conidiation, primary and secondary metabolism,including mycotoxin biosynthesis. In this review we summarise some recent findings on the effect of specific lightwavelength and intensity on mycotoxin biosynthesis in the main toxigenic fungal genera. We describe the molecularmechanism underlying light perception and its involvement in the regulation of secondary metabolism, focusing onVeA, global regulator in Aspergillus nidulans, and the White-Collar proteins, key components of light response inNeurospora crassa. Light of specific wavelength and intensity exerts different effects both on growth and on toxinproduction depending on the fungal genus. In Penicillium spp. red (627 nm) and blue wavelengths (455-470 nm)reduce ochratoxin A (OTA) biosynthesis by modulating the level of expression of the ochratoxin polyketide synthase.Furthermore a mutual regulation between citrinin and OTA production is reported in Penicillium toxigenic species.In Aspergillus spp. the effect of light treatment is strongly dependent on the species and culture conditions. Royalblue wavelength (455 nm) of high intensity (1,700 Lux) is capable of completely inhibit fungal growth and OTAproduction in Aspergillus stenyii and Penicillum verrucosum. In Fusarium spp. the effect of light exposure is lesseffective; mycotoxin-producing species, such as Fusarium verticillioides and Fusarium proliferatum, grow betterunder light conditions, and fumonisin production increased. This review provides a comprehensive picture onlight regulation of mycotoxin biosynthesis and discusses possible new applications of this resource in food safety.

Light is an important environmental signal which influence many different physiological responses such as pigmentation, sexual development, asexual conidiation, the circadian clock and secondary metabolism. Our studies about light regulation on metabolic pathways in fungi proved that light of specific wavelength and intensity influences fungal growth and mycotoxin production.In Fusarium proliferatum light generally had a positive influence on growth, with a mean increase of the grow rate of about 40% under light exposure in comparison to the dark. Wavelengths from both sides of the spectrum, from long (627 nm) to short wavelength (470-455 nm) had a stimulating effect on fumonisin biosynthesis: fumonisins B1 and B2 production increased of about 40 fold under red, 35 fold under blue, 20 fold under royal blue, 10 fold under green, 5 fold under yellow and 3 fold under white light in comparison to the dark incubation. In Fusarium verticillioides wavelengths from red (627 nm) to blue (470-455 nm) stimulated the growth and increased the fumonisin production, by up to 150 % over dark incubation. On the contrary if the intensity of the 455 nm blue light increased from 200 to 1700 lx, the fumonisins biosynthesis decreased. Incubation under a short wave blue light (390 nm) showed reduced fungal growth and fumonisin production by up to 85 %. White pulsing light had no effect on growth but reduced fumonisin production to half of what observed during dark incubation. In both these Fusarium spp Real time reverse transcriptase analysis showed a correlation between fumonisins biosynthesis and FUM gene expression.These findings demonstrated a role of light on the growth and the modulation of fumonisin biosynthesis and provide new information on the physiology of important toxigenic pathogens.

In this work the authors analyzed sixty italian strains of the edible mushroom "cardoncello" (Pleurotus eryngii) belonging to the varieties eryngii and ferulae by sequencing two housekeeping genes (ef1-a and rpb2) and evaluating the activity of peroxidase, superoxide dismutase and catalase, in order to find some molecular markers for the traceability of "cardoncello". Sequence analysis showed the presence of Snps variety-specific (eryngii and ferulae) in both genes useful for the development of molecular identification tools. none correlation between enzymatic analysis and analyzed varieties was observed.

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.

The human health benefits of fruits and vegetables are ascribed to theirphytochemical content, such as carotenoids and polyphenolics. In the search forantioxidative chemicals from native fruits of the Puglia region of Italy, Prunus cerasusL., an acidic cherry widely used for culinary purposes, and Prunus mahaleb L., a treespecies commonly used as rootstock in cherry crop, were studied. The P. mahalebfruits have a high content of organic acids, fructose and vitamin C, but are notconsumed fresh because of a bitter and sour taste. In this work we obtained the 1HNMR spectra of the two species and from the comparison of these spectra, we foundthat P. mahaleb fruits have an higher concentration of phenolic compounds, such asflavonoids, and organic acids, in comparison to P. cerasus fruits. The same resultswere obtained when we focused on anthocyanins. In this study we identified thesignals of anthocyanin protons in 1H NMR spectra of a mixture of compounds inaqueous extracts of both P. cerasus and P. mahaleb fruits but the latter species showeda higher concentration and a larger number of these compounds. This metabolomicanalysis gave us the data to scientifically revalue traditionally-used plants like P.mahaleb and to identify the potential as source of biofunctional compounds to be usedin food and/or pharmaceutical industry. Moreover, in this study, NMR spectroscopycoupled with multivariate data analysis was applied to Prunus metabolomics in orderto investigate the botanical origins of Prunus cerasus and to identify the compoundsresponsible for differentiation of these two species of Prunus (cerasus and mahaleb)and of two cultivars of Prunus cerasus ('Montmorency' and 'Marasca di Zara').

A two-year study on the pathosystem F. verticillioides-maizewas conducted speculating on both in vitro and in planta perspectives.The former studied the effects of temperature (T) andwater activity (aw) on fumonisin B (FBs) production and expressionof FUM2 and FUM21 genes in ITEM 10027 and 1744 F. verticillioidesstrains grown up to 21 days. The latter monitoredwhich genes were differentially expressed in resistant and susceptiblemaize lines after 2-3 days infection by ITEM 1744. The invitro study showed that ITEM 10027 was the highest FBs producer,with predominance of FB1. The maximum level of FB wasregistered after 21 days in both strains. FUM2 and FUM21 wereexpressed in all studied conditions, with a 10X difference betweenthe former and the latter. The peak of transcription levelwas reached after 14 days of incubation for both FUM genes. Nodata were collected on cultures grown at fixed aw=0.900 becausethe fungus did not grow at all temperatures tested till 21 days ofincubation. The in planta study showed that resistant lines testedwere poorly infected by ITEM 1744. Genes differentially expressedwere divided into 11 functional categories and nearly10% was assigned to the class "cell rescue, defence and virulence".Most of the pathogenesis-related genes were differentiallyactivated after fungal infection in relation to the resistance levelof maize genotypes. In the resistant kernels, defence-relatedgenes provided basal protection against the fungus, while in thesusceptible kernels, the same genes were induced specifically afterpathogen attack.

Fungal biodiversity is one of the most important contributors to the occurrence and severity of mycotoxincontamination of crop plants. Phenotypic and metabolic plasticity has enabled mycotoxigenic fungi to colonizea broad range of agriculturally important crops and to adapt to a range of environmental conditions.New mycotoxin-commodity combinations provide evidence for the ability of fungi to adapt to changing conditionsand the emergence of genotypes that confer enhanced aggressiveness toward plants and/or alteredmycotoxin production profiles. Perhaps the most important contributor to qualitative differences in mycotoxinproduction among fungi is variation in mycotoxin biosynthetic genes. Molecular genetic and biochemicalanalyses of toxigenic fungi have elucidated specific differences in biosynthetic genes that are responsible forintra- and inter-specific differences in mycotoxin production. For Aspergillus and Fusarium, the mycotoxigenicgenera of greatest concern, variation in biosynthetic genes responsible for production of individual families ofmycotoxins appears to be the result of evolutionary adaptation. Examples of such variation have been reportedfor: a) aflatoxin biosynthetic genes in Aspergillus flavus and Aspergillus parasiticus; b) trichothecene biosyntheticgeneswithin and among Fusarium species; and c) fumonisin biosynthetic genes in Aspergillus and Fusarium species.Understanding the variation in these biosynthetic genes and the basis for variation inmycotoxin productionis important for accurate assessment of the risks that fungi pose to food safety and for prevention of mycotoxincontamination of crops in the field and in storage.

The Pleurotus eryngii species complex is an economically important group which includes several closely related varieties, whose genetic discrimination is still not clear. One hundred and ten Italian strains of Pleurotus eryngii belonging to the varieties elaeoselini, eryngii, ferulae and thapsiae and P. nebrodensis were analysed by sequencing two housekeeping genes (ef1-a and rpb2), in order to find molecular markers for the identification of different varieties. Sequence analysis of partial ef1-a and rpb2 genes, allowed identification of some conserved nucleotide positions within each variety but variable among var. elaeoselini, var. eryngii, var. ferulae var. thapsiae and P. nebrodensis, allowing their discrimination. Phylogenetic analysis from the data of the two genes data set showed that var. elaeoselini, var. thapsiae, var. ferulae and var. eryngii are closely related to each other, and confirm P. nebrodensis as a separate clade.

Dried vine fruits may be heavily colonized by Aspergillus species. The molecular biodiversity of an Aspergillus population (234 strains) isolated from dried vine fruit samples of worldwide origin were analyzed by investigating four housekeeping gene loci (calmodulin, beta-tubulin, elongation factor 1-alpha, RPB2). Aspergillus Sect. Nigri was dominant and the strains were identified as A. tubingensis (138), A. awamori (38), A. carbonarius (27),A. uvarum (16) and A. niger (11). Four Aspergillus flavus strains were also identified from Chilean raisins. Two clusters closely related to the A. tubingensis species with a significant bootstrap (60% and 99%) were identified as distinct populations. Among the four loci, RPB2 showed the highest genetic variability. This is the first complete study on the worldwide distribution of black Aspergilli occurring on dried vine fruits identified by a molecular approach.

Living biomass on the planet is represented for 50% by microorganisms that provide an important source of genetic information for both molecular biology and biotechnology. Fungi play a major bio-regulatory role in natural ecosystems and represent an extraordinary source of new compoundsof great ecological relevance. In particular, the toxigenic fungi (TF) produce a large seriesof secondary metabolites (SMs),that mayaccumulatein final products of agro-food plants. These compoundspossess a wide range of biological activities with a high impact on plant, human and animal health.An important category of these specialised metabolites are formed by mycotoxins, due to the detrimental effect on other organisms, including humans and animals. Therefore, incorrect identification of TFwill havenegative consequences on the accurate evaluation ofexposure risk for the consumption of contaminated food. Currently, many studies on the characterization of TFat genetic and biochemical level generate a huge amount of oftenunrelated and not well organized data. On the other hand, the scientific community can take advantagefrom both a more rational organization of such data and extensivesharing of the organismsthat produce these compounds. To further progress of the general knowledge on TF, fundamental steps are needed including reduction of overlaps and optimization of the efforts at global level.Tofacilitatemerging of informationand preservenatural biodiversity, important objects should be pursued such as: i) identification and characterization of TFs using a standard and polyphasic approach; ii) organization and sharingof data; iii) deposition of strains in well recognized Culture Collections.The Horizon 2020 EU project MycoKey(Grant 678781) aims to reduce mycotoxin contaminationinfood and feed crops. Among the activities inthe project, great attention is madeon thecarefuldeposition of toxigenic fungi and the harmonization ofrelevant information related to TFsand (changes in) their global occurrence. Datasets include genomic sequences and SMs annotations, DNA sequences, SMs profiles, and metadata on their geographic occurrence and ecological niches. Sharing knowledge and biological materials willultimately provide an effective contribution to mycotoxin risk management.

MycoKey, an EU-funded Horizon 2020 project, includes a series of "Roundtable Discussions" to gather information on trending research areas in the field of mycotoxicology. This paper includes summaries of the Roundtable Discussions on Chemical Detection and Monitoring of mycotoxins and on the role of genetics and biodiversity in mycotoxin production. Discussions were managed by using the nominal group discussion technique, which generates numerous ideas and provides a ranking for those identified as the most important. Four questions were posed for each research area, as well as two questions that were common to both discussions. Test kits, usually antibody based, were one major focus of the discussions at the Chemical Detection and Monitoring roundtable because of their many favorable features, e.g., cost, speed and ease of use. The second area of focus for this roundtable was multi-mycotoxin detection protocols and the challenges still to be met to enable these protocols to become methods of choice for regulated mycotoxins. For the genetic and biodiversity group, both the depth and the breadth of trending research areas were notable. For some areas, e.g., microbiome studies, the suggested research questions were primarily of a descriptive nature. In other areas, multiple experimental approaches, e.g., transcriptomics, proteomics, RNAi and gene deletions, are needed to understand the regulation of toxin production and mechanisms underlying successful biological controls. Answers to the research questions will provide starting points for developing acceptable prevention and remediation processes. Forging a partnership between scientists and appropriately-placed communications experts was recognized by both groups as an essential step to communicating risks, while retaining overall confidence in the safety of the food supply and the integrity of the food production chain.

Worldwide mycotoxins contamination has a significant impact on animal and human health, and leads to economic losses accounted for billions of dollars annually. Since the application of pre- and post- harvest strategies, including chemical or physical removal, are not sufficiently effective, biological transformation is considered the most promising yet challenging approach to reduce mycotoxins accumulation. Although several microorganisms were reported to degrade mycotoxins, only a few enzymes have been identified, purified and characterized for this activity. This review focuses on the biotransformation of mycotoxins performed with purified enzymes isolated from bacteria, fungi and plants, whose activity was validated in in vitro and in vivo assays, including patented ones and commercial preparations. Furthermore, we will present some applications for detoxifying enzymes in food, feed, biogas and biofuel industries, describing their limitation and potentialities.

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

Aspergillus section Nigri populations isolated from seven growing regions from Argentina were characterizedby sequencing in order to identify species responsible for production of ochratoxin A (OTA) and fumonisins(FBs). Sequences of genes encoding calmodulin, ?-tubulin, the second largest subunit of RNA polymerase IIand translation elongation factor 1 alpha were analysed. The phylogenetic analysis showed the presence of sixlineages: A. carbonarius, A. tubingensis, A. niger, A. japonicus, A. homomorphus and A. foetidus grouped in fourmajor clusters. The molecular tools used allowed the identification for the first time of A. homomorphus fromvineyards. OTA production confirmed the importance of A. carbonarius as the main ochratoxigenic speciesisolated and, to a variable degree, of A. niger and A. tubingensis, which were by far the most commonlyoccurring species on grapes in Argentina. The only strains able to produce OTA and fumonisins (B2-B4) belongto the A. niger cluster.

The healthy consumers make a strong pressure to natural products that can prevent the chronic diseases and improve the general health status, and therefore an important aspect that have to be considered is the safe level of the nutraceuticals. This study reports the occurrence of Ochratoxin A (OTA) and associated fungal contamination in 35 samples of dried vine fruits imported in the European community potentially used for the development of new nutraceutical supplements. High pressure liquid chromatography analysis identified 18 samples as contaminated by OTA with an average level of 2.6 ?g/kg. OTA was measured in 4 samples of currants (mean value of 6.6 ?g/kg) and 13 samples of raisins (mean value of 1.4 ?g/kg). In one sample of currants and one of raisins from Turkey OTA exceeded the limits set by European Commission of 10 ?g/kg, being contaminated with 12.61 and 15.99 ?g/kg, respectively. All the positive samples were confirmed by Orbitrap Q Exactive through their molecular weight and the corresponding fragmentation. The worldwide consumption of dried vine fruits contributed to OTA exposure in several group of consumers. In particular, considering the potential nutraceutical approach, this consumption may be represent a severe risk for healthy consumers that consider these products like healthy and salutistic for their contents in antioxidants, flavonoids, and polyphenols. Data reported in this study confirmed the need to regularly monitor mycotoxin levels in these food products and optimize the process of fruits drying in order to reduce the development of toxigenic molds.

Fusarium verticillioides and Fusarium proliferatum are the mainsource of fumonisins, a group of mycotoxins that can contaminate maize-based food and feed and cause diseases in humans and animals.We analyzed the effect of temperature (15-35°C), water activity(aw: 0.999-0.93), salinity (0-125 g/l NaCl), pH (5-8) and light ofdifferent wavelength (650-390 nm) on the growth, the productionof fumonisins B (FB) and the expression of FUM1 and FUM21. ForF. verticillioides the highest growth rate was measured at 25°C, awof 0.998-0.99, 0-25 g/l of NaCl and white light. Optimal conditionsfor fumonisin production were 30°C, aw of 0.99, 25 g/l of NaCl,pH 5, red and blue light. For Fusarium proliferatum the highestgrowth rate was measured at 25°C, aw of 0.99 and 0-25 g/l of NaCl,pH 7 and green light. Optimal conditions for fumonisin productionwere 25°C, aw of 0.998, 0 g/l of NaCl, pH 6, red and bluelight. F. verticillioides showed a better adaptability compared toF. proliferatum and was able to produce moderate levels of fumonisinsunder a wide range of conditions. FUM gene expression notalways mirrored FB production, indicating a post-transcriptionalmechanism regulationg fumonisin production. The study of theeffect of different environmental conditions on toxin productionshould provide information that can be used to develop strategiesto minimize the risk.

Fusarium verticillioides is the main source of fumonisins, a group of mycotoxins that can contaminate maize-based food and feed and cause diseases in humans and animals. The study of the effect of different environmental conditions on toxin production should provide information that can be used to develop strategies to minimize the risk. This study analysed the effect of temperature (15°C-35°C), water activity (a (w): 0.999-0.93), salinity (0-125 g l(-1) NaCl) and pH (5-8) on the growth and production of fumonisins B(1) (FB1), B(2) (FB2) and B(3) (FB3) and the expression of FUM1 and FUM21 in F. verticillioides. The highest growth rate was measured at 25°C, a (w) of 0.998-0.99 and 0-25 g l(-1) of NaCl. Optimal conditions for fumonisin production were 30°C, a (w) of 0.99, 25 g l(-1) of NaCl and pH 5; nevertheless, the strain showed a good adaptability and was able to produce moderate levels of fumonisins under a wide range of conditions. Gene expression mirrored fumonisin production profile under all conditions with the exception of temperature: FUM1 and FUM21 expression was highest at 15°C, while maximum fumonisin production was at 30°C. These data indicate that a post-transcriptional regulation mechanism could account for the different optimal temperatures for FUM gene expression and fumonisin production.

L'aflatossina M1 (AFM1) è il principale metabolita derivante dall'idrossilazionedell'aflatossina B1 (AFB1) presente nel latte di animali alimentati con mangimi contaminati da AFB1 ed è classificato nel gruppo 2B, potenzialmente cancerogeno per l'uomo, dall'AgenziaInternazionale per la Ricerca sul Cancro (IARC). Il livello limite della AFM1 nel latte crudo,trattato termicamente e destinato alla produzione di prodotti a base di latte è fissato a50ng/kg dal regolamento europeo numero 1881 del 2006. Essendo resistente ai comunitrattamenti dell'industria alimentare, la presenza di AFM1 è documentata in tutti i prodottidella filiera lattiero casearia, inclusi yogurt e formaggi, e rappresenta un serio pericolo perla salute. Lo sviluppo di metodi per la riduzione della contaminazione di aflatossine è untema cruciale e attuale, ed è complicato dalla necessità di preservare le qualità organolettichee nutrizionali della matrice trattata. In questo lavoro è stata valutata la capacità degradativadi una laccasi da Pleurotus eryngii verso l'AFM1, sia in buffer che in latte, ed il suo effettosulla componente proteica di questa matrice al fine di verificarne l'applicazione per ilmiglioramento della sicurezza di prodotti lattiero caseari. La riduzione di AFM1 in bufferdi sodio acetato pH 6.5 1mM, a 25°C, è di ca 50% dopo 1h di incubazione e risulta completadopo 72h. Simili risultati sono stati ottenuti in latte, sebbene la cinetica di degradazioneabbia registrato un rallentamento nelle prime tre ore di trattamento. L'analisi dei patternproteici in SDS-PAGE ha evidenziato una riduzione nell'intensità delle bande di ? e ?caseine, di ?-lattoglobulina e sieroalbumina bovina, contemporaneamente alla comparsadi aggregati proteici di peso molecolare superiore ai 200kDa. I dati presentati dimostranoil potenziale applicativo della laccasi per lo sviluppo di metodologie green di degradazionedi AFM1 in prodotti a base di latte e per applicazioni tecnologiche volte al miglioramentodella reologia e alla riduzione della componente allergenica in prodotti lattierocaseari.Parole chiave: sicurezza, latte, laccasi, aflatossina M1, cross-link di proteine, reologia dellatte, allergeni

Biodiversity research concerns with data coming from many different domains (e.g., Biology, Geography, Evolutionary Studies, Genomics, Taxonomy, Environmental Sciences, etc.) which need to be integrated for leading to valuable Biodiversity knowledge. Collecting and integrating data from so many heterogeneous resources is not a trivial task. Data are extremely scattered, heterogeneous in format and purpose, and protected in repositories of several research institutes. Driven by the widely diffused trend of the web of sharing information through aggregation of people with the same interests (social networks), and by the new type of database architecture defined as dynamic distributed federated database, we are proposing a new paradigm of data integration in the Biodiversity domain. Here we present a new approach for the development of a Knowledge Base aiming to the collection, integration and analysis of biodiversity data implemented as a product of the MBLab project.

Fusarium genus is able to produce several metabolites including the emerging mycotoxins beauvericin (BEA) and enniatins (ENs). Due to their ionophoric property, BEA and ENs exert many biological properties, including antimicrobial, insecticidal, and cytotoxic activity in human cell lines, so that they are recently proposed as novel anticancer drugs. BEA and ENs are cyclic hexadepsipeptides with an alternating sequence of three N-metyl-L-amino acids and three D-?-hydroxyisovaleric acids; in BEA the amino acid residues are aromatic N-metyl-pheylalanines, whereas in ENs the amino acid residues are aliphatic N-metyl-valine, -leucine or -isoleucine. Both mycotoxins are synthesized by the multifunctional enzyme enniatin synthetase (ESYN1) representing hybrid system of peptide synthetase and S-adenosyl-L-methionine-dependent N-methyltransferase. Several Fusarium species have been reported to produce ENs, BEA or both, and our hypothesis is that the different production profile depends on the esyn1 sequence. Our aim was to investigate this relation by mean of a bioinformatics approach, based on structural investigations.The esyn1 sequences of 18 Fusarium isolates belonging to 8 different species were extracted from published and unpublished genomes by BLASTN search using as query the available sequences of Fusarium scirpi and Fusarium proliferatum. The protein sequences were predicted using the Sequence Translation tools of the EMBOSS Programs (EMBL-EBI), manually curated using exon/intron boundary predictions from SpliceView (http://bioinfo4.itb.cnr.it/), and confirmed by sequencing the RT-PCR products.The selected sequences have been processed by the web server RaptorX to obtain structural predictions by homology modelling and threading methods. While the full sequences have been submitted for prediction, it has been accomplished only for part of the whole enzyme. In particular, the region containing the N-methyltransferase activity has been not completely structured. A comprehensive analysis of the predicted structures has allowed identifying common structural domains, which have been compared by considering their dihedral backbone angles and by using the tool T-PAD, developed to characterize the protein flexibility and identify hot spot residues responsible for hinge motions. The residue-by-residue flexibility profiles have been analysed by multivariate analysis, to produce a classification by Principal Component Analysis, followed by hierarchical clustering.We have thus identified the conserved structural regions and the pivotal residues responsible for the structural variability. The common domains for each of the 18 esyn1 sequences have been grouped in clusters, and the residues responsible for the classification have been singled out. Overall, we have achieved a comprehensive view of the structural features of the analysed esyn1 sequences, where the structural variability has been related to the sequence variability, and interpreted in terms

Ochratoxin A (OTA) is a nephrotoxic and potentially carcinogenic mycotoxin produced by several species of Aspergillus and Penicillium, contaminating grapes, wine and a variety of food products.We recently isolated from OTA contaminated soil vineyard a novel free-livingstrain of Acinetobacter sp. neg1, ITEM 17016, able to degrade OTA into the nontoxic catabolic product OTalpha (OT?). Biochemical studies suggested that the degradation reaction proceeds via peptide bond hydrolysis with phenylalanine (Phe) release. In order to identify genes responsible for OTA degradationwe performed a differential gene expression analysis of ITEM 17016 grown in the presence or absence of the toxin. Among the differentially expressed genes, 6 peptidases up-regulated at 6 hours were identified. The degrading activity of the carboxypeptidase PJ_1540 was confirmed in vitro in a heterologous system. The enrichment analysis for Gene Ontology terms confirmed that OTA degradation proceeds through peptidase activities and revealed the over-representation of pathways related to Phe catabolism. These results indicate that Phe may represent an energy source for this Acinetobacter sp. neg1strain and that OTA degrading reaction triggers the modulation of further catabolic activities.