Aflatoxin contamination of tree nuts is a growing concern for pistachio producing countries. Development of competitive exclusion strategies through application of atoxigenic Aspergillus flavus isolates is a highly effective route of natural aflatoxin mitigation. Aflatoxin assays conducted on a high number of native A. flavus isolates are a first step to identify potential biological control isolates. Many cultural methods for the rapid and visual identification of atoxigenic A. flavus isolates have been described. The current study identified atoxigenic A. flavus isolates from Iranian pistachio orchards using and contrasting cultural, analytical and molecular methods. Ammonium vapour (AV) and fluorescence detection (FD), two rapid aflatoxin assays, were directly compared using various media preparations to screen 524 A. flavus isolates obtained from Iranian pistachio orchards. Percentages of false negatives were high using FD assays for all media preparations ranging from 13 to 15 %. This in contrast to AV assays. Here incidences of false negatives ranged from 0 % (using coconut agar medium) to 7.2 % (using potato dextrose agar). Aflatoxin-producing ability of all isolates was further confirmed using thin layer- and high-performance liquid chromatography. Sixty three atoxigenic A. flavus isolates were identified as atoxigenic in all assays. For these isolates, five loci across the aflatoxin biosynthesis cluster pathway were compared to identify genetic defects explaining atoxigenicity. Genetic deletions in at least one of five loci in the aflatoxin biosynthesis pathway were found for 97 % of isolates. Frequencies of atoxigenic strains ranged from 7.1 to 37.5 % with the lowest incidence detected in the Kerman province. Proper identification of atoxigenic isolates is considered a first step in the development of biological control strategies. Ability of identified isolates to competitively exclude aflatoxin-producing fungi has to be further investigated. © 2014 Koninklijke Nederlandse Planteziektenkundige Vereniging.

Fusarium species, particularly Fusarium graminearum and F culmorum, are the main cause of trichothecene type B contamination in cereals. Data on the distribution of Fusarium trichothecene genotypes in cereals in Europe are scattered in time and space. Furthermore, a common core set of related variables (sampling method, host cultivar, previous crop, etc.) that would allow more effective analysis of factors influencing the spatial and temporal population distribution, is lacking. Consequently, based on the available data, it is difficult to identify factors influencing chemotype distribution and spread at the European level. Here we describe the results of a collaborative integrated work which aims (1) to characterize the trichothecene genotypes of strains from three Fusarium species, collected over the period 2000-2013 and (2) to enhance the standardization of epidemiological data collection. Information on host plant, country of origin, sampling location, year of sampling and previous crop of 1147 F graminearurn, 479 F culmorum, and 3 F cortaderiae strains obtained from 17 European countries was compiled and a map of trichothecene type B genotype distribution was plotted for each species. All information on the strains was collected in a freely accessible and updatable database (www.catalogueeu.luxmcc.lu), which will serve as a starting point for epidemiological analysis of potential spatial and temporal trichothecene genotype shifts in Europe. The analysis of the currently available European dataset showed that in F. grarninearum, the predominant genotype was 15-acetyldeoxynivalenol (15-ADON) (82.9%), followed by 3-acetyldeoxynivalenol (3-ADON) (13.6%), and nivalenol (NIV) (3.5%). In F culmorum, the prevalent genotype was 3-ADON (59.9%), while the NIV genotype accounted for the remaining 40.1%. Both, geographical and temporal patterns of trichothecene genotypes distribution were identified.

A new solid-phase extraction (SPE) pretreatment method using a home-made polyvinylpolypyrrolidone-florisil (PVPP-F) column was developed for the analysis of patulin in apple and hawthorn products in China. Fifty samples (25 apple juices, 12 apple jams, and 13 hawthorn juices) were prepared using the new method and then analyzed by high performance liquid chromatography with diode array detection (HPLC-DAD) on an Agela Venusil MP C(18) reversed-phase column (4.6 mm × 250 mm, 5 ?m). The cleanup results for all samples using home-made PVPP-F column were compared with those obtained using a MycoSep®228 AflaPat column. The correlation coefficient R (0.9998) fulfilled the requirement of linearity for patulin in the concentration range of 2.5-250 ?g/kg. The limits of detection (LODs) and quantification (LOQs) of patulin were 3.99 and 9.64 ?g/kg for PVPP-F column, and 3.56 and 8.07 ?g/kg for MycoSep®228 AflaPat column, respectively. Samples were spiked with patulin at levels ranging from 25 to 250 ?g/kg, and recoveries using PVPP-F and MycoSep®228 AflaPat columns were in the range of 81.9-100.9% and 86.4-103.9%, respectively. Naturally occurring patulin was found in 2 of 25 apple juice samples (8.0%) and 1 of 13 hawthorn juice samples (7.7%) at concentrations ranging from 12.26 to 36.81 ?g/kg. The positive results were further confirmed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS).

DNA-based phylogenetic analyses have resolved the fungal genus Fusarium into multiple species complexes. TheF. incarnatum-equiseti species complex (FIESC) includes fusaria associated with several diseases of agriculturallyimportant crops, including cereals. Although members of FIESC are considered to be only moderately aggressive,they are able to produce a diversity of mycotoxins, including trichothecenes, which can accumulate to harmfullevels in cereals. High levels of cryptic speciation have been detected within the FIESC. As a result, it is often necessaryto use approaches other than morphological characterization to distinguish species. In the current study,we used a polyphasic approach to characterize a collection of 69 FIESC isolates recovered from cereals in Europe,Turkey, and North America. In a species phylogeny inferred from nucleotide sequences from four housekeepinggenes, 65 of the isolateswere resolvedwithin the Equiseti clade of the FIESC, and four isolateswere resolvedwithinthe Incarnatum clade. Seven isolates were resolved as a genealogically exclusive lineage, designated here asFIESC 31. Phylogenies based on nucleotide sequences of trichothecene biosynthetic genes and MALDI-TOF MS(Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) were largely concordant withphylogeny inferred from the housekeeping gene. Finally, Liquid Chromatography (Time-Of-Flight) Mass Spectrometry[LC-(TOF-)MS(/MS)] revealed variability in mycotoxin production profiles among the different phylogeneticspecies investigated in this study.

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

Biological degradation of mycotoxins is an emerging strategy for detoxification of agricultural commodities. In particular, enzymatic degradation of aflatoxin B1 (AFB1), the most harmful among the mycotoxin known which may occur as contaminant of most of food and feed, has lately raised considerable scientific interest. Ligninolytic enzymes, such as laccase and peroxidise, from white-rot fungi have been proven to be able to break the highly stable molecule of AFB1. However, the high cost of production and purification of these enzymes have limited their implementation into practical technologies aimed at reduction of aflatoxin contamination in the food and feed chains. Among the white-rot fungi there are also cultivable edible mushrooms, such as Pleurotus spp. Every year tons of spent mushroom substrate (SMS) are produced as a by-product of mushroom cultivation and disposed at a cost for farmers. However, SMS may still be a source of bioactive compounds, including ligninolytic enzymes potentially useful for degradation of aflatoxin. We investigated the AFB1-degradative activity of a crude extract (CE) of SMS and undertook a study for characterization of enzyme content and stability of the extracts. CE of SMS was obtained by an extraction buffer (sodium phosphate buffer 0.1 M, pH 7.3) and the extract was incubated with 1 ?g/ml of AFB1 at 25 °C, under continuous shaking at 120 rpm for 1, 3 and 7 days; then the aflatoxin content was determined by ultra performance liquid chromatography (UPLC). After 1 day of incubation, the CE was able to degrade more than 50% of AFB1 and after 3 and 7 days of incubation the percentage of degradation reached the values of 75% and 90%, respectively. The CE contained a high level of laccase activity, quantified in 4 Units per gram of SMS dry weight (U/g DW) and low level of Mn-peroxidase (0.4 U/g DW), as determined by spectrophotometric assays. The enzymatic activity of the CE had its optimum at temperature ranging between 5 and 25 °C and at pH 4, and it was stable at +4 °C for about 60 days. Heat treatment at 100 °C for 10 minutes completely destroyed the degradative activity of CE, and freeze drying resulted in a decrease by 35% of the laccase activity. Based on these preliminary results, SMS proved to be a suitable source of aflatoxin-degrading enzymes and the use of SMS and/or their CE for detoxification of aflatoxin-contaminated commodities, particularly those intended for feed, appears as a coinceivable technology for aflatoxin-free feed production. Further research is needed to improve the stability of SMS extracts and to implement their use in the pipeline of feed processing.

The presence of virus and bacteria in the airways of subjects with asthma is common and seems to be associated with a deterioration due to the disease. The microbiologic study of airways in asthma is foreseen by guidelines with induced sputum that is often ineffective and contraindicated in severe asthma. Aim: To analyze the fungal microbiome in the exhaled breath condensate (EBC) of subjects with asthma by evaluating a possible correlation with anthropometric and asthma severity data. Methods: We enrolled 47 consecutive subjects with asthma (28 with atopic asthma and 19 with nonatopic asthma) and 20 controls. Enrolled subjects underwent EBC and sputum collection. Fungal microbiome was assessed by culture on EBC and sputum samples by using Czapek yeast extract agar. Results: A fungal colonization in the EBC of 70% of enrolled subjects with asthma was detected (none detected in the controls). An overlap of fungal microbiome in EBC and sputum was observed (100% of overlap). Fungal colonization was higher in subjects without atopic, obesity, and severe and uncontrolled asthma. Conclusion: When considering the high morbidity and mortality of patients with severe asthma in whom we found an important fungal airways colonization, we support the use of the analysis of exhaled fungal microbiome in these subjects.

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

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.

Airways of lung cancer patients are often colonized by fungi. Some of these colonizing fungi, under particular conditions, produce cancerogenic mycotoxins. Given the recent interest in the infective origin of lung cancer, with this preliminary study we aim to give our small contribution to this field of research by analysing the fungal microbiome of the exhaled breath condensate of lung cancer patients from Puglia, a region of Italy.Methods:We enrolled 43 lung cancer patients and 21 healthy subjects that underwent exhaled breath condensate and bronchial brushing collection. The fungal incidence and nature of sample collected were analysed by using a selected media for Aspergillus species.Results:For the first time we were able to analyse the fungal microbioma of the exhaled breath condensate. 27.9% of lung cancer patients showed a presence of Aspergillus niger, or A. ochraceus or Penicillium ssp. while none of the healthy subjects did so.Conclusion:The results confirmed the high percentage of fungal colonization of the airways of lung cancer patients from Puglia, suggesting the need to conduct further analyses in this field in order to evaluate the exact pathogenetic role of these fungi in lung cancer as well as to propose efficient, empirical therapy. © 2014 Carpagnano et al.; licensee BioMed Central Ltd.

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).

This work is the first large-scale study on vineyard-associated yeast strains from Apulia (Southern Italy).Yeasts were identified by Internal Transcribed Spacer (ITS) ribotyping and bioinformatic analysis. Thepolymorphism of interdelta elements was used to differentiate Saccharomyces cerevisiae strains. Twentydifferent species belonging to 9 genera were identified. Predominant on the grape surface wereMetschnikowia pulcherrima, Hanseniaspora uvarum and Aureobasidium pullulans, whereas M. pulcherrimaand H. uvarum were dominant in the early fermentation stage. A total of 692 S. cerevisiae isolates wereidentified and a number of S. cerevisiae strains, ranging from 26 to 55, was detected in each of the eightfermentations. The strains were tested for biogenic amines (BAs) production, either in synthetic media orgrape must. Two Pichia manshurica, an Issatchenkia terricola and a M. pulcherrima strains were able toproduce histamine and cadaverine, during must fermentation. The production of BAs in wine must wasdifferent than that observed in the synthetic medium. This feature indicate the importance of an "ingrape must" assessment of BAs producing yeast. Overall, our results suggest the importance of microbiologicalcontrol during wine-making to reduce the potential health risk for consumer represented bythese spoilage yeasts.

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

Fungal biodiversity is one of the most important contributors to the occurrence and severity of mycotoxin contamination of crop plants. Phenotypic and metabolic plasticity has enabled mycotoxigenic fungi (MF) to colonize a 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 conditions and the emergence of genotypes that confer enhanced aggressiveness toward plants and/or altered mycotoxin production profiles.Among diseases caused by MF, the most important are the result of attacks carried out by species complexes. Examples of these diseases are the Fusarium ear rot of maize, caused by species of the Fusarium fujikuroi species complex; Fusarium head blight of small-grain cereals (e.g. wheat, barley, and oat) caused by Fusarium graminearum species complex and a number of other Fusarium species; black point of wheat kernels caused by Alternaria alternata species complex and related species; grape rot caused by the black Aspergilli. Thus, the ability of various toxigenic species within the complexes to produce different classes of secondary metabolites, combined with their ability to coexist in the same host or/and occur in quick succession have allowed these complexes to become "invincible armadas" against many plants. Plant infections by MF can occur at all developmental stages, from germinating seeds to mature vegetative tissues, depending on the host plant and MF species involved. Therefore, since most toxigenic fungi have specific mycotoxin profiles, early and accurate identification of the species occurring in the plants at every step of their growth is critical to predict the potential toxicological risk to which plants are exposed and to prevent toxins entering the food chain. Moreover, the great biodiversity of MF species/strains is clearly the main factor responsible for the multi-toxin contamination risk in food commodities due to the co-occurrence of groups of toxigenic fungi genetically closely related or distant on the same crop. In addition, a great contribution to qualitative differences in mycotoxin production among fungi is variation in mycotoxin biosynthetic genes. We will report in the presentation main genetic, biochemical and chemical investigations carried out at the ISPA in order to: i) establish phylogenetic relatedness among the main species belonging to Alternaria, Aspergillus and Fusarium genera occurring on agro-food host plant products by using a multi-locus approach; ii) elucidate some specific differences in biosynthetic genes that are responsible for intra- and inter-specific differences in fumonisin and trichothecene production in Aspergillus and Fusarium, respectively; iii) develop rapid, easy and robust multi-mycotoxin detection methods for an accurate and reliable assessment of the risks related to the mycotoxin contamination of food products in the field.

The variation in the coding capacity within Oenococcus oeni can have a significant impact on wine quality. The detection of several genes involved in important metabolic pathways (i.e. citrate, sulphur and arginine metabolisms) was performed on 10 indigenous O. oeni strains from Negroamaro wine, a red table wine (Apulia, Italy). These strains were selected from 95 isolates, collected during spontaneous malolactic fermentation, according to the results of an Amplified Fragment Length Polymorphism (AFLP) analysis. A total of 16 genes were screened, most (11) of which had never previously been assayed on O. oeni. All strains possessed 10 genes encoding enzymes such as malolactic enzyme (mleA), esterase (estA), citrate lyase (citD, citE and citF), citrate transporter (maeP), alpha-acetolactate decarboxylase (alsD), alpha-acetolactate synthase (alsS), S-adenosylmethionine synthase (metK) and cystathionine beta-lyase (metC) and resulted negative in the detection of genes encoding cystathionine gamma-lyase (metB), ornithine transcarbamylase (arcB) and carbamate kinase (arcC). The sequence of PCR fragments of 11 genes of a representative strain (ITEM 15929) was compared to those of three reference O. oeni strains. The indigenous strain was phylogenetically more similar to PSU-1 and ATCC BAA1163 than AWRI B429. This study describes new genetic markers useful for detecting the genetic potential of O. oeni strains to contribute to aroma production and for investigating the population structure of the species. (C) 2014 Elsevier Ltd. All rights reserved.

In this study, the naturally debittered table olives cv Bella di Cerignola were studied in order to (i) characterizetheir phenolic composition; (ii) evaluate the polyphenols bioaccessibility; (iii) assess their absorption and transport, acrossCaco2/TC7. LC-MS/MS analysis has confirmed the presence of hydroxytyrosol acetate, caffeoyl-6?-secologanoside, andcomselogoside. In vitro bioaccessibility ranged from 7% of luteolin to 100% of tyrosol, highlighting the flavonoids sensitivity tothe digestive conditions. The Caco2/TC7 polyphenols accumulation was rapid (60 min) with an efficiency of 0.89%; the overallbioavailability was 1.86% (120 min), with hydroxytyrosol and tyrosol the highest bioavailables, followed by verbascoside andluteolin. In the cells and basolateral side, caffeic and coumaric acids metabolites, probably derived from esterase activities, weredetected. In conclusion, the naturally debittered table olives cv Bella di Cerignola can be considered as a source of bioaccessible,absorbable, and bioavailable polyphenols that, for their potential health promoting effect, permit inclusion of table olives as afunctional food suitable for a balanced diet.

The main objective of the present study was the characterization of phenolic compounds from "Bella di Cerignola" table olive cultivar, one of the most important variety in Apulia (Southern Italy), that received the "Protected Denomination of Origin" (DOP) in 2000 by EU. In addition, the assessment of bioaccessibility of these polyphenols was investigated by using in vitro gastro- intestinal digestion model1. Methodologies: After extraction, the phenolic concentration was determined by HPLC according to Lattanzio 1982(2). Moreover, the olives were subjected to successive gastric and pancreatic digestion, following the method of Versantvoort et al. (2005)1 .The polyphenols were identified by HPLC-DAD and LC-HRMS analysis in order to assess their bioaccessibility. Results and conclusion: The main polyphenols identified were: hydroxytyrosol, tyrosol, caffeic acid, verbascoside, isoverbascoside, caffeoyl-6-secologanoside, comselogoside, luteolin. Interesting is the presence of comselogoside and caffeoyl-6-secologanoside, already identified in OMWW3 and olive fruits4 . After in vitro digestion process, all total phenols identified were bioaccessible, apart luteolin that was absent in the digestive fraction. In particular, the bio-accessibilities were: 84.2% for hydroxytyrosol, 100% for tyrosol and caffeic acid, 47 % for verbascoside, 75% for isoverbascoside, 77% for caffeoyl-6-secologanoside, 81% for comselogoside. Regarding tyrosol, caffeic acid and isoverbascoside, their amount, in the chyme fraction, was higher than no digested olives, probably for hydrolysis and isomerizations phenomena occurred in the gastro-intestinal conditions. In conclusion, the data obtained provide preliminary insight on the potential for bioavailability of the polyphenols present in a complex matrix such as "Bella di Cerignola" cultivar.

Aflatoxin B1 (AFB1) is the most harmful mycotoxin that occurs as natural contaminant of agricultural commodities, particularly maize. Practical solutions for detoxification of contaminated staples and reduction of agricultural wastes are scarce. We investigated the capability of the white-rot and edible fungus Plerotus eryngii (king oyster mushroom) to degrade AFB1 both in vitro and in a laboratory-scale mushroom cultivation, using a substrate similar to that routinely used in mushroom farms. In malt extract broth, degradation of AFB1 (500 ng/mL) by nine isolates of P. eryngii ranged from 81 to 99% after 10 days growth, and reached 100% for all isolates after 30 days. The growth of P. eryngii on solid medium (malt extract-agar, MEA) was significantly reduced at concentrations of AFB1 500 ng/mL or higher. However, the addition of 5% wheat straw to the culture medium increased the tolerance of P. eryngii to AFB1 and no inhibition was observed at a AFB1 content of 500 ng/mL; degradation of AFB1 in MEA supplemented with 5% wheat straw and 2.5% (w/v) maize flour was 71-94% after 30 days of growth. Further, AFB1 degradation by P. eryngii strain ITEM 13681 was tested in a laboratory-scale mushroom cultivation. The mushroom growth medium contained 25% (w/w) of maize spiked with AFB1 to the final content of 128 ?g/kg. Pleurotus eryngii degraded up to 86% of the AFB1 in 28 days, with no significant reduction of either biological efficiency or mushroom yield. Neither the biomass produced on the mushroom substrate nor the mature basidiocarps contained detectable levels of AFB1 or its metabolite aflatoxicol, thus ruling out the translocation of these toxins through the fungal thallus. These findings make a contribution towards the development of a novel technology for remediation of AFB1- contaminated corn through the exploitation of the degradative capability of P. eryngii and its bioconversion into high nutritional value material intended for feed production.

Leaves of three different sweet basil (Ocimum basilicum L.) cultivars (Italico a foglia larga, Cammeo, andItaliano classico) packed in macro-perforated polyethylene bags were stored at chilling (4 C) or nonchillingtemperature (12 C) for 9 days. During storage, visual quality, physiological (respiration rate,ethylene production, ammonium content) and chemical (antioxidant activity, total polyphenols andpolyphenol profile) parameters were measured. Detached leaves stored at chilling temperature showedvisual symptoms related to chilling injury, while ethylene production and ammonium content resultedassociated to cultivar sensibility to damage at low temperature. Storage at 4 C caused a depletion inpolyphenols content and antioxidant capability, which was preserved at 12 C. Regarding the polyphenolsprofile, stressful storage conditions did not enhance the phenolic metabolism. However, leavesstored at 12 C did not loss a significant amount of metabolites respect to fresh leaves, suggesting the possibilityto extend the storability after the expiration date, for a possible recovery of bioactive compounds.

Pasta represents one of the most well-known products all over the world. It is a traditional food of Mediterrean diet and it is generally manufactured with durum wheat, that confers the excellent rheologicalproperties of dough, the superior color, the appearance and the cooking quality compared to other flours. The World Health Organization (WHO) and Food and Drug Administration (FDA) consider spaghetti pastaa good vehicle for the addition of nutrients and consumers are increasingly interested in foods containing health promoting ingredients. In this contest, the objective of the present study was to characterize thebioactive components, such as carotenoids, triterpenic acids and polyphenols, present in durum wheat semolina spaghetti enriched with an olive oil patè coming from olive oil processing chain, and dried inorder to obtain a flour (OPF) suitable for the pasta process. In particular, OPF was added to the pasta dough at 10% and 15% (w/w). Finally, spaghetti with 10% OPF, considered acceptable to the sensory panel test, were characterized for their bioactive components. Further, their bioaccessibility, after simulated gastro-intestinal digestion, was also assessed. Results showed that 10% OPF addition enriched the spaghetti in ?-tocopherol, ?- and ?-carotene, maslinic and eonolic acids. The ratio of polyunsaturated (PUFA) to saturated fatty acids (SFA) resulted higher (1.16) than spaghetti used as control (0.69). The total polyphenols content (free, conjugated and bound) in dry spaghetti samples increased of about 3 times respect to the control. In particular, the amount of total free phenolics in the enriched sample, was almost 50 times higher respect to the control spaghetti, with tyrosol and oleuropein as the most abundant. In addition, it is interesting to underline that the enriched spaghetti showed an high amount of flavonoids, such as apigenin, luteolin and quercetin already present in OPF. The phenolic acids were mainly present in the conjugated and bound fractions with the highest amount in the spaghetti with 10% OPF. Finally, the bioactive components showed a good level of biaccessibility, although during the cooking process, the 50% was naturally lost.

The prickly pear cactus, Opuntia ficus-indica (OFI) is a tropical or subtropical plant originally grown in South America; its cladodes represent a by-product that generally is used for animal feed. Moreover, they are rich in pectin, mucilage and minerals, vitamins and antioxidants and the use as food medicine and cosmetic ingredients, could be proposed. In the present study, the characterization of bioactive compounds (polyphenols, sugars and minerals) in dehydrated OFI cladodes were performed. Further, antioxidant activity and polyphenols/minerals stability after simulated gastro-intestinal digestion, were also evaluated.

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.

Fumonisin contamination of maize is considered a serious problem in most maize-growing regions of the world,due to the widespread occurrence of these mycotoxins and their association with toxicosis in livestock andhumans. Fumonisins are produced primarily by species of Fusarium that are common in maize grain, but alsoby some species of Aspergillus sect. Nigri, which can also occur on maize kernels as opportunistic pathogens. Understandingthe origin of fumonisin contamination in maize is a key component in developing effectivemanagementstrategies. Although some fungi in Aspergillus sect. Nigri are known to produce fumonisins, little is knownabout the species which are common in maize and whether they make a measurable contribution to fumonisincontamination ofmaize grain. In thiswork,we evaluated populations of Aspergillus sect. Nigri isolated frommaizein USA and Italy, focusing on analysis of housekeeping genes, the fum8 gene and in vitro capability of producingfumonisins. DNA sequencing was used to identify Aspergillus strains belonging to sect. Nigri, in order to comparespecies composition between the two populations, which might influence specificmycotoxicological risks. Combinedbeta-tubulin/calmodulin sequences were used to genetically characterize 300 strains (199 from Italy and101 fromUSA)which grouped into 4 clades: Aspergilluswelwitschiae (syn. Aspergillus awamori, 14.7%), Aspergillustubingensis (37.0%) and Aspergillus niger group 1 (6.7%) and group 2 (41.3%). Only one strain was identified as Aspergilluscarbonarius. Species composition differed between the two populations; A. niger predominated amongthe USA isolates (69%), but comprised a smaller percentage (38%) of Italian isolates. Conversely, A. tubingensisand A. welwitschiae occurred at higher frequencies in the Italian population (42% and 20%, respectively) than inthe USA population (27% and 5%). The evaluation of FB2 production on CY20S agar revealed 118 FB2 producingand 84 non-producing strains distributed among the clades: A. welwitschiae, A. niger group 1 and A. niger group2, confirming the potential of Aspergillus sect. Nigri species to contribute to total fumonisin contamination ofmaize. A higher percentage of A. niger isolates (72.0%) produced FB2 compared to A. welwitschiae (36.6%). Thepercentage of FB2-producing A. niger strains was similar in the USA and Italian populations; however, the predominanceof A. niger in the USA population suggests a higher potential for fumonisin production. Some strainswith fum8 present in the genome did not produce FB2 in vitro, confirming the ineffectiveness of fum8 presence asa predictor of FB2 production.

In green leafy vegetables, the retention of green colour is one of the most generally used index to evaluatethe overall quality and freshness and it is associated to total chlorophyll content.Destructive chemical techniques and non-destructive chlorophyll meters represent the state-of-the-artmethods to accomplish such critical task. The former are effective and robust but also expensive and timeconsuming. The latter are cheaper and faster but exhibit lower reliability, require the probe to touch theleaves and heavily depend on the positions chosen for sampling the leaf's surface. In this paper, a newapproach to non-destructively predict total chlorophyll content of fresh-cut rocket leaves without contactis proposed. Fresh-cut rocket leaves were analysed for total chlorophyll content by spectrophotometerand SPAD-502 (used as reference values) and acquired by a computer vision system using a machinelearningmodel (Random Forest Regression) to predict total chlorophyll content. Finally, the trainedand validated model will be used for on-line prediction of total chlorophyll content of unseen freshcutrocket leaves. The proposed system can match the physical and timing constraints of a real industrialproduction line and its performance (R2 = 0.90), measured on the case study of fresh-cut rocket leaves,outperformed the results of the SPAD chlorophyll meter (R2 = 0.79).

CONTINNOVA project was funded by Apulia Regional government within the Call on Technological Cluster for Innovations. It started in November 2015 and will be concluded within November 2017. The main objective will be to create a refrigerated isothermal container equipped with innovative technologies, including advanced sensors for the control of the atmosphere composition, during the transport of fresh fruit and vegetables. The final goal is to ensure the quality and safety of fruit and vegetables products of regional Apulia exports along the distribution chain. The partnership consists of the IFAC spa (leader), the companies TERA and Ditro, the Department of Agro-Food Sciences, Food and Environment (SAFE) of the University of Foggia, the Departments of Mechanics, Mathematics and Management (DMMM) and Civil Engineering, Environmental, Land, Construction and Chemistry (DICATECh) of the Polytechnic of Bari, Spin-Off INGENIUM, Apulia Food Fingerprint Lab Network and the Institutes of CNR ISSIA (Institute of Intelligent Systems Research For Automation) and ISPA (Institute of Sciences of Food Production), the latter with the role of Scientific Adviser. The industrial companies designed and are realizing the container, firstly as Prototype (in scale) and then in real dimension. While, the scientific partners researched the best atmosphere composition and/or atmosphere pre-treatment to be implemented in the Container in order to transport in optimal condition table grape and fresh artichoke. In the last work package of the project, the innovative container will be validated during the simulation of an experimental transport of table grape. Finally, the innovative container will be made available to Association of producer and exporter of horticultural products (APEO) to test its performance in preserving quality and extending shelf-life of the vegetables transported.

Forty-five samples of a landrace of sweet pepper (Capsicum annuum) widely cultivated in Basilicata (Italy) werescreened for 17 mycotoxins and potential toxigenic fungal species. Two different LC-MS/MS methods were usedfor the determination of aflatoxins, ochratoxin A (OTA), Fusarium mycotoxins zearalenone (ZEA), fumonisins (FB1and FB2), nivalenol (NIV), deoxynivalenol (DON), T-2 and HT-2 toxins and Alternaria mycotoxins altenuene (ALT),alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TTX) and tenuazonic acid (TeA). Frequencyof potential toxigenic fungal species occurrence was: 87% Aspergillus Sect. Nigri; 58% Aspergillus Sect. Flavi; 38%Aspergillus Sect. Circumdati; 42% Alternaria spp.; 33% Penicillium spp. and 20% Fusarium spp. Frequency ofmycotoxin occurrence and mean of positives were: 51% OTA, 29.5 µg/kg, 5 samples above the EU limit of 20 µg/kg;31% aflatoxins, 12.8 µg/kg, two samples above the EU limit of 5 µg/kg for aflatoxin B1; 91% ZEA, 1.4 µg/kg; 78%FB2, 7.6 µg/kg; 58% FB1, 22.8 µg/kg; 38% NIV, 39.5 µg/kg; 36% DON, 6.9 µg/kg; 20% T-2 toxin, 5.6 µg/kg and 22%HT-2 toxin, 13.8 µg/kg. For the Alternaria mycotoxins, 100% of samples contained TeA, 4817.9 µg/kg; 93% TTX,29.7 µg/kg; 56% AOH, 114.4 µg/kg; 33% AME, 13.0 µg/kg and 9% ALT, 61.7 µg/kg. Co-occurrence of mycotoxinsin each sample ranged from 2 to 16 mycotoxins (mean 7). No statistical correlation was found between mouldsand their mycotoxins occurrence. Within the four groups of peppers collected herein (fresh, dried, grounded andfried) higher percentages of contamination and mycotoxin levels were measured in grounded peppers, whereasmuch lower values were observed for fried peppers. The high percentages of positive samples and the high levelsof some mycotoxins observed in this study confirm the susceptibility of peppers to mycotoxin contamination andclaims for an improvement of the conditions used during production and drying process.

Aspergillus niger is a significant component of the fungal community on grapes. The mycotoxinfumonisin B2 (FB2) was recently detected in grape must and wine as well as in cultures of someA. niger strains isolated from grapes and raisins. This study examined 48 strains of Aspergillus sectionNigri for the presence of the fumonisin biosynthetic gene fum8 in relation to FB2 production. The fum8gene was detected in only 11 A. niger strains, 9 of which also produced FB2. Maximum parsimonyanalysis based on the calmodulin gene sequence indicated that the presence/absence of fum8 is notcorrelated with the phylogenetic relationship of the isolates. This is the first report correlating thepresence of a fumonisin biosynthetic gene with fumonisin production in A. niger from an important foodcrop. The results suggest that the absence of FB2 production in grape isolates of A. niger can resultfrom the absence of at least one gene essential for production.

Fumonisin B1 (FB1) is among the most common mycotoxins found in maize kernels and maize productsworldwide. The microbiological process of detoxification and transformation of toxic organic pollutants is a promisingmethod for foodstuffs decontamination. Some basidiomycetes, such as the Pleurotus eryngii species complex,include several important commercial edible varieties that can detoxify polycyclic organic compounds and a rangeof wastes and pollutants. We investigated the potential role of P. eryngii, one of the most consumed mushrooms, inthe decontamination of FB1 in maize. In addition, selected antioxidant enzymes, (soluble peroxidase (POD), catalase(CAT) and ascorbate peroxidase), primarily involved in control of cell hydrogen peroxide levels, and lignindegradation, were analyzed, to evaluate their contributions to the molecular mechanisms of FB1 by P. eryngii. FB1decontamination by P. eryngii and involvement of CAT and POD enzymes in the control of toxic decontaminationlevels of H2O2 were demonstrated. A consistent reduction of FB1 was observed at different incubation times. Theaverage decrease levels of FB1, with respect to the control cultures, ranged from 45 to 61% (RSD < 15%). This studyis a possible eco-friendly approach to reducing this mycotoxin in the feed supply chains.

An unprecedented, environmentally friendly, and faster method for the determination ofOchratoxin A (OTA) (a mycotoxin produced by several species of Aspergillus and Penicillium andlargely widespread in nature, in wheat and derived products) has, for the first time, been set up andvalidated using choline chloride (ChCl)-based deep eutectic solvents (DESs) (e.g., ChCl/glycerol(1:2) and ChCl/ urea (1:2) up to 40% (w/w) water) as privileged, green, and biodegradable extractionsolvents. This also reduces worker exposure to toxic chemicals. Results are comparable to thoseobtained using conventional, hazardous and volatile organic solvents (VOCs) typical of the standardand official methods. OTA recovery from spiked durum wheat samples, in particular, was to up to89% versus 93% using the traditional acetonitrile-water mixture with a repeatability of the results(RSDr) of 7%. Compatibility of the DES mixture with the antibodies of the immunoaffinity columnwas excellent as it was able to retain up to 96% of the OTA. Recovery and repeatability for durumwheat, bread crumbs, and biscuits proved to be within the specifications required by the currentEuropean Commission (EC) regulation. Good results in terms of accuracy and precision wereachieved with mean recoveries between 70% (durum wheat) and 88% (bread crumbs) and an RSDrbetween 2% (biscuits) and 7% (bread).

Traditional Chinese medicinal plants (TCMPs), commonly used as spices, additives or foods, are also widely used in China to prevent and cure human disease. Due to their provenance, TCMPs may be contaminated by various fungal species, including ochratoxigenic fungi, during growth, collection, transportation and, especially, storage. A reliable method for the determination of ochratoxin A (OTA) in TCMPs of different origins was developed to monitor OTA levels in China. Analyses involved the extraction of OTA by methanol/water, immunoaffinity column (IAC) clean-up and HPLC quantification with fluorescence detection (FLD). Positive results were further confirmed by LC-ESI-MS/MS. The limit of detection (LOD) was 0.3 mu g kg-1, based on a signal-to-noise ratio of 3 : 1. Among the total of 57 TCMPs samples collected from six different areas, 31 were visibly moldy due to inappropriate storage; 26 sample, purchased from local TCMPs markets, were not visibly moldy. The results showed that 23 of the visibly moldy samples and two of the not visibly moldy were contaminated with OTA at levels ranging 1.2-158.7 and 2.5-5.6 mu g kg-1, respectively. This is the first report of the natural occurrence of OTA in TCMPs

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

Development, validation and one-site testing of immunoassays for rapid mycotoxin detection is one of the priority tasks of the MycoKey project (http://www.mycokey.eu/), an EU multidisciplinary project, aimed to develop smart solutions to reduce the major occurring mycotoxins in economically important food and feed chains. In this framework, a multiplex dipstick immunoassay based method for the simultaneous quantitative determination of major Fusarium toxins, namely deoxynivalenol (DON), zearalenone (ZEA), and fumonisins (FUM) in cereals (wheat, barley and maize) is under development. The dipstick format is based on an indirect competitive approach. The DON/ZEA/FUM prototype is based on a nitrocellulose lateral flow device using a reader to enable quantitative determination of mycotoxin contamination in cereal extracts. Three test lines (mycotoxin-BSA conjugates) and one control line are located on the strip membrane, whereas labeled antibodies were freeze-dried within a microwell. The prototype strip test development included the following steps: definition of coating process and detection reagent formulation parameters; definition of assay parameters like incubation time, migration time, volume sampling to reach minimum performance requested in term of LoD (limit of detection), LoQ (limit of quantification); development of a sample preparation protocol allowing satisfactory mycotoxin recoveries. The total time of analysis is 25 minutes including pre analytical treatment.Preliminary results showed the DON/ZEA/FUM prototype to be compatible with the minimum acceptable performances, and to be suitable for its application to the analysis of real samples containing the target mycotoxins at levels close to EU regulatory levels.

Fusarium ear rot is one of the most important diseases of maize, that is of concern because Fusarium verticillioides produces the mycotoxins known as fumonisins. F. verticillioides can be transmitted either through infected silks or seed-to-kernel. In order to better understand the virulence of F. verticillioides, the effect of the fungus on the defense systems was investigated both in immature kernels and in seedlings. The molecular mechanisms involved in compatible and incompatible responses were also studied. Gene expression data were obtained from microarray hybridizations, comparing healthy and infected kernels of resistant and susceptible maize inbreds 48 h after infection with a fumonisin-producing strain of F. verticillioides. A total of 739 transcripts were differentially expressed between the two inbred lines at one time point after infection. Among all the differentially regulated genes, 7.3% of encoded proteins play a role in cell rescue and defense. The qRT-PCR analysis confirmed that most of the defense genes had already been transcribed before infection occurred in the maize-resistant line. The study was extended to the analysis of enzymes involved in removing reactive oxygen species, namely ascorbate peroxidase, catalase, total peroxidase and superoxide dismutase. In resistant seedlings, before infection, ascorbate peroxidase and superoxide dismutase enzyme activities were higher than in the susceptible ones and, 5 days after treatment, they remained unchanged. On the other hand, in the susceptible seedlings, except for superoxide dismutase, all enzymes assayed were activated after pathogen attack. These results support our previous findings of a basal defense response provided by maize genotypes resistant against F. verticillioides infection, both in kernels and seedlings.

Introduction Food authenticity aims to guarantee product safety, transparency and protection of consumers' health. In order to safeguard food authenticity, a variety of analytical techniques have been investigated some of which are routinely used. If on one hand some methodologies typically based on MS detection revealed successful for the identification of selected molecules as suitable markers for species identification, on the other hand the untargeted methods utilizing MS detection appear not deeply investigated in this specific field. In the present communication, a non targeted approach based on DART ionization coupled to High Resolution Mass Spectrometry was described and applied for the discrimination of two types of salmons -farmed versus wild type-, belonging to the Salmo salar species and collected in Canada. In particular, the apolarfractions were analysed and the final data were further subjected to multivariate statistical analysis to highlight eventual differences between bothdata-set. ExperimentalDifferent fish homogenates were prepared from 26 canadian wild type salmons (CND-WT) and25 canadian farmed salmons (CND-F). Aliquots of 2.5 g of each fish homogenate were added with 5 mL of refrigerated methanol and 5 mL of refrigerated chloroform and stirred for 15 min at R.T.After decantation for 2 hours at 4°Cthe lower apolar fraction was withdrawn and first evaporated under nitrogen flow and then resuspended in an equal volume of isopropanol before HRMS analysis. Analysis were performed by DART ionization (IonSense) coupled to HRMS using an Exactive(TM) (Thermo Fisher Scientific) system in negative ion mode by setting the gas temperature 150°C. The total peak lists of all ions generated from each analysis was converted in .csv format and processed for the multivariate statistical analysis by using the free online software MetaboAnalyst 3.0. ResultsAfter duly inspecting the HRMS spectra referred to the analysis of the apolar fraction, it appears that the ion m/z 281.247 is the most intense in CND-F samples despite the ion m/z 255.232 instead dominating the CND-WT spectra. By entering the m/z values into the LipidMaps database, the ion species were likely attributed to thestearic acid (m/z 281.247) and palmitic acid (m/z 255.232) suggesting their possible role as potential biomarkers indicator of the type of capture. By applying the multivariate statistical analysis e.g. the Principal Component Analysis(PCA) on the list of ionsa good separation between the two fish groups (farmed vs wild type) was foundwith PC1 and PC2 that explained respectively 68.5% and 23.3% of the total variance.ConclusionsIon differences between the CND-F and CND-WT samples werehighlightedby applying DART-HRMS analysis followed by multivariate statistical analysis on the whole dataset. In particular, PCA resultsshowed a good separation of both salmon groups. In future, a supervisioned approach might be envisaged to further improve such separation between b

Lentil (Lens culinaris Medik.) is the fourth most important pulse crop in the world after bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), and chickpea (Cicer arietinum L.). Canada is the world's largest exporter of lentils, while in Italy lentils are a minor legume and can be found in restricted areas. However, Italian lentils present unique and characteristic qualities giving them a higher value, so that many of them have obtained international and national marks linked to their geographical origins, such as "protected geographical indication" (PGI), "traditional food products" (PAT) and Slow Food Presidium. For these reasons, there is a growing demand for analytical methods able to certify the declared geographical origin of lentils, in order to protect consumers and producers from fraud and unfair competition. In the present work, non-targeted 1H-NMR fingerprinting, in combination with different multivariate statistical analysis techniques, was used to classify lentils according to their geographical origin. In particular, 85 lentil samples from two different countries, i.e. Italy and Canada, were collected from retail markets and analysed by using an optimized 1H-NMR protocol. Principal component analysis showed partial grouping of samples on the basis of origin with overlapping zones. Therefore, a class-modeling technique, Soft Independent Modelling of Class Analogy (SIMCA), and three discriminant techniques, such as k - Nearest Neighbor (k-NN), Linear Discriminant Analysis (LDA), Partial Least Squares - Discriminant Analysis (PLS-DA), were used and the performances of the resulting models were compared. The best average recognition and cross-validation prediction abilities, 100% and 93.7% respectively, were obtained by the LDA model, performed on a set of 20 principal components previously selected by a stepwise decorrelation procedure. The other models, except the SIMCA one, also showed good performances (above 90%). All tested statistical models were validated by evaluating the prediction abilities on an external set of lentil samples. LDA model showed the best results with an external prediction ability of 100%, but also the other models showed remarkable performances (above or near 90%).These findings demonstrated the suitability of the methods developed to discriminate geographical origin of lentils and confirmed the applicability of the NMR data, in combination with chemometrics, to solve geographic origin issues of foodstuffs.

Lentil (Lens culinaris Medik.) is the fourth most important pulse crop in the world after bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), and chickpea (Cicer arietinum L.). Canada is the world's largest exporter of lentils, while in Italy lentils are a minor legume and can be found in restricted areas. However, Italian lentils present unique and characteristic qualities giving them a higher value, so that many of them have obtained international and national marks linked to their geographical origins, such as "protected geographical indication" (PGI), "traditional food products" (PAT) and Slow Food Presidium. For these reasons, there is a growing demand for analytical methods able to certify the declared geographical origin of lentils, in order to protect consumers and producers from fraud and unfair competition. In the present work, non-targeted 1H-NMR fingerprinting, in combination with different multivariate statistical analysis techniques, was used to classify lentils according to their geographical origin. In particular, 85 lentil samples from two different countries, i.e. Italy and Canada, were collected from retail markets and analysed by using an optimized 1H-NMR protocol. Principal component analysis showed partial grouping of samples on the basis of origin with overlapping zones. Therefore, two class-modeling techniques such as Soft Independent Modelling of Class Analogy (SIMCA) and UNEQual dispersed classes (UNEQ) and three discriminant techniques, such as k - Nearest Neighbor (k-NN), Linear Discriminant Analysis (LDA), Partial Least Squares - Discriminant Analysis (PLS-DA), were used and the performances of the resulting models were compared. The best average recognition and cross-validation prediction abilities, 100% and 93.7% respectively, were obtained by the LDA model, performed on a set of 20 principal components previously selected by a stepwise decorrelation procedure. The other models, except the SIMCA one, also showed good performances (above 90%). All tested statistical models were validated by evaluating the prediction abilities on an external set of lentil samples. LDA model showed the best results with an external prediction ability of 100%, but also the other models showed remarkable performances (above or near 90%).These findings demonstrated the suitability of the methods developed to discriminate geographical origin of lentils and confirmed the applicability of the NMR data, in combination with chemometrics, to solve geographic origin issues of foodstuffs.

Mycotoxin risk in the grape product chain is primarily due to ochratoxin A (OTA) occurrence in wine and dried vine fruits. Aspergillus carbonarius and the A. niger group are the main agents of Aspergillus bunch rot of grape, and they, especially A. carbonarius, are responsible for OTA contamination worldwide. Fumonisin B2 (FB2) represents an additional potential mycotoxin risk in the grape-wine product chain and A. niger/A. awamori were recently reported as the FB2 producers in grapes. A deeper understanding of the species diversity of black Aspergilli, together with specific knowledge of their ecology and epidemiology, can help to predict their occurrence. From this perspective several studies have been done regarding prevention and control of black Aspergilli and reduction of mycotoxin risk at all stages, from vineyard management to wine-making procedures. In this review a comprehensive overview of all these aspects is presented.

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.

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.

We analyzed the microbiological quality and safety of raw milk from vending machine and assessed the evolution of its microbiota during the refrigeration at 7°C. We also monitored the survival of E. coli O157:H7 strain artificially added to the raw milk during the refrigeration and investigated the possibility to use the probiotic Lactobacillus rhamnosus GG strain to control pathogenic and spoilage bacteria in raw milk. Total aerobic mesophilic microbiota of raw milk exceeded the limit allowed by the current European legislation and increased of about two log units after 48 hr of refrigeration at 7°C. The spiked E. coli O157:H7 strain multiplied slowly in the refrigerated raw milk increasing of about 1 log unit, whereas the probiotic strain was able to slightly lower the populations of Pseudomonas spp., Listeria spp. and Staphylococcus aureus but was unable to inhibit the growth of E. coli O157:H7. Practical applications: This study allowed us to confirm the microbiological threat associated to the direct consumption of raw milk. Ad hoc strategies to improve the overall microbiological quality of raw milk from vending machines within the whole chain (from farm to consumption) are needed. A protective and probiotic adjunct able to control spoilage and pathogenic bacteria in raw milk with an adequate microbiological quality (at least compliant with the current legislation) may be a valuable mitigation strategy, but probiotic strains more effective than L. rhamnosus GG should be used.

Table olives are a typical food of the Mediterranean diet and an important source of phenolic compoundswith high biological potential for human health. Their concentrations (?1-2% of FW) confer to table olivesantioxidant, anti-inflammatory, and antitumoral properties. The polyphenols content and composition in table olives can be affected by several factors, such as cultivars, climate, fruits ripeness, and, mainly, theprocessing methods. Among the de-bittering processes, the Greek method represents a spontaneous fermentation procedure that is driven by a mixed population of microorganisms, mainly consisting of yeastsand lactic acid bacteria (LAB). In this work, the effects of fermentation by autochthonous yeast and LAB starters on phenolics composition of Apulian table olives, Bella di Cerignola (BDC), Termite di Bitetto(TDB) and Cellina di Nardò (CEL) were studied in comparison with the commercial products. The samples were characterized by HPLC-DAD for their polyphenols composition; 18 compounds were identified andthe cultivar related effect was highlighted. The main identified phenolics were hydroxytyrosol, tyrosol, verbascoside and luteolin, followed by hydroxytyrosol-acetate detected in BDC and cyanidine-3-glucosideand quercetin in CEL. Further, the fermentation using selected LAB and yeasts influenced differently the composition and amount of polyphenols of the three cultivars, in particular the BDC amount was doubledcompared with the commercial sample. Instead, for TDB and CEL, the treatment did not influence the polyphenols composition. It is noteworthy that the use autochthonous microbial starter to drive table olivesfermentation process allows to maintain stable or increases polyphenols concentration in fermented table olives, significantly reducing the time necessary for de-bittering and improving organoleptic and sensorycharacteristics of the final product. Scavenger capacity in both DPPH and CAA assays, assessed the highest antioxidant effect for CEL with starters (21.7 mg Trolox eq/g FW; 8.5 ?mol hydroxytyrosol eq/100g FW).Moreover, the polyphenols were highly in vitro bioaccessible (>60%), although modifications in their profile, probably for combined effect of environment and microorganisms, were noted.Finally, fermented table olives are excellent source of health promoting compounds. Indeed, hydroxytyrosol and tyrosol are almost 8 times more than in olive oil for which a nutritional EU claim (No433/2012 of 23 May 2012) that attributes the protective effect from oxidative stress by polyphenols on blood lipids, has been established.

Contamination of vineyards from black Aspergilli is a well-known condition that cause the accumulation of ochratoxin A (OTA) in grapes and derived products. This contamination is strongly related to climatic conditions, geographical regions (South Mediterranean climate is highly conducive), grape varieties, damage by insects, although, great variations may occur from one year to another. Among the black Aspergilli commonly found in infected grapes, Aspergillus carbonarius is considered the main responsible of OTA contamination, with A. niger at secondary extent. To minimise the black Aspergilli infection and limit OTA concentrations in grapes, several strategies are commonly adopted, including the implementations of good agricultural practices and the use of pesticides and fungicides. These strategies are essential to manage the problem, but since they are insufficient when extremely favourable condition occurs in the vineyard, new strategies, aimed to reduce OTA risk in vineyards, are necessary. In this respect, implementation of electrolysed oxidising water (EOW) in agriculture has arising during the last decade as an interesting alternative to replace or limit the use of chemicals. The efficacy of EOW was also demonstrated in post-harvest for reduction of gray mold and brown rot on surfaces of peaches and grapes. In this study, we screened for the first time the efficacy of EOW generated by EVA System® 100 apparatus (Industrie De Nora S.p.A., Milan, Italy) at different concentrations of free chlorine (ranging from 0.0125 to 0.4 g/L) on conidial germination and growth of A. carbonarius and A. niger. A good fungicidal activity was achieved after 2-10 minutes treatment with EOW containing 0.4-0.2 g/L of free chlorine, although A. carbonarius conidia were more resistant to EOW than A. niger conidia. Then EOW at 0.4 g/L free chlorine was tested on detached berries of Primitivo and Aglianico wine grape varieties that were singularly infected with A. niger and A. carbonarius. Treatments with Switch (cyprodinil and fludioxonil) at 0.8 g/L, a fungicide regularly used in vineyard against black Aspergilli, were used as positive controls. Percentage of infected berries, McKinney index, and OTA concentrations were used to evaluate the efficacy of the EOW treatment. On Aglianico grape berries EOW and Switch produced an almost complete reduction on percentage of infection, McKinney index and OTA concentration compared to the control. On Primitivo grape berries EOW treatment reduced more than half the percentage of infections and McKinney index for both A. carbonarius and A. niger, although Switch showed a better performance. A significant reduction of OTA concentration was observed for EOW and Switch treatments. These results evidence for the first time that EOW is effective to reduce black Aspergilli inoculum and OTA contamination on grape berries.

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.

Aflatoxin B1 (AFB1) is a major threat to human and animal health, due to its potent hepatotoxic, carcinogenic and mutagenic effects. Removal or inactivation of aflatoxin in food and feedstuff is difficult. Chemical and physical methods have been found to be effective in detoxification of AFB1 from various materials but their use in the practice is limited, due to safety issues and possible loss of nutritional value of the treated commodities. In the feed industry a proposed technology for detoxification of feedstuff is the dietary supplementation of inorganic or organic materials able to adsorb mycotoxins. Once the mycotoxin has been bound, absorption in the digestive tract of animals is strongly reduced. Some mineral adsorbents such as aluminosilicates, bentonites and activated carbons have showed good results but have some drawbacks, such as the negative impact on the nutritional quality of decontaminated feed. For this reason, interest in the use of natural microbial adsorbents has increased. In this study we investigated the capability of ground not-viable mycelium of the edible and cultivated mushroom Pleurotus eryngii to bind AFB1. P. eryngii strain ITEM 13681 was cultured on malt-extract broth for 20 days at 28 °C in the dark. For preparation of the biosorbent, the mycelium was harvested, autoclaved, lyophilized and finely ground to particles of size <= 500 µm. One hundred milligrams of the biosorbent were suspended in 5 mL of phosphate-buffer saline (PBS) pH 7 containing 2000 ng AFB1, and incubated overnight at 25 °C in a rotary shaker at 250 rpm. Then, the mycelium and the supernatant were separated by centrifugation and both the phases were analyzed for AFB1 content. Sixty-five percent of AFB1 was detected in the supernatant, while the mycelium was able to retain 6 ng of AFB1 per milligram of dry mycelium (35%). The effect of mycelium dosage was studied by testing different amounts of biomass (25, 50, 75, 100, 150, 200 mg/mL) in both acetate buffer (pH = 5) and PBS (pH = 7) containing 0.5 µg/mL AFB1. The suspensions were shaken for 90 minutes at 250 rpm, at 25 and 37 °C. Then the samples were centrifuged at 13000 rpm for 10 minutes and the pellets were washed three times and analyzed by UPLC/FLD. The efficiency of adsorption (%A) was calculated using following equation: %A = [(Ci - Cf) / Ci] x 100; where Ci was the initial and the Cf final concentration (supernatant plus washing solution) of mycotoxin. The biosorbtion showed to be both dosage- and temperature-dependant. In acetate buffer the mycelium adsorbed 44±4% at the dosage of 200 mg and no significant adsorption at 25 mg. In PBS absorption at 37 °C ranged from 7±5% at 25 mg of mycelium to 64±6% at 200 mg, and at 25 °C it ranged from 3±0 % at 25 mg of mycelium to 26±3% at 200 mg. Our results envisage a possible use of P. eryngii as a cheap and effective biosorbent for AFB1. This mechanism of AFB1-binding by P. eryngii mycelium is herein demonstrated for the first time.

A pressing necessity of the Apulian wine industry is to being able to pilot and to control the wine production to obtain wines with peculiar characteristics and with respect of the typicality guaranteed by the denominations of origin. The employment of selected autochthonous yeast strains would be a potent instrument to improve the organoleptic and sensory characteristics of typical regional wines. In fact, indigenous yeasts are better adapted to a specific must and therefore they are able to exalt the peculiarities of the derived wine. The present work described the genetic diversity of autochthonous Saccharomyces cerevisiae strains derived from natural must fermentations of an important Apulian grape cultivar, denoted as “Primitivo”. The yeast strains showing the best technological and oenological properties were selected and their fermentative performances were assayed by either laboratory tests and industrial scale fermentations. Two autochthonous yeast strains showed to be good candidates as industrial starter cultures, since they dominated the fermentation process and produced wines characterized by peculiar oenological and organoleptic features, that were judged very pleasant by a panel of winemakers.

The effects of fermentation by autochthonous microbial starters on phenolics composition of Apulian table olives, Bella di Cerignola (BDC), Termite di Bitetto (TDB) and Cellina di Nardò (CEL) were studied, highlighting also the cultivars influence. In BDC with starter, polyphenols amount doubled compared with commercial sample, while in TDB and CEL, phenolics remain almost unchanged. The main phenolics were hydroxytyrosol, tyrosol, verbascoside and luteolin, followed by hydroxytyrosol-acetate detected in BDC and cyanidine-3-glucoside and quercetin in CEL. Scavenger capacity in both DPPH and CAA assays, assessed the highest antioxidant effect for CEL with starters (21.7 mg Trolox eq/g FW; 8.5 ?mol hydroxytyrosol eq/100g FW). The polyphenols were highly in vitro bioaccessible (>60%), although modifications in their profile, probably for combined effect of environment and microorganisms, were noted. Finally, fermented table olives are excellent source of health promoting compounds, since hydroxytyrosol and tyrosol are almost 8 times more than in olive oil.

Fusarium toxins, a group of mycotoxins, can be produced by Fusarium fungi under temperate climatic conditions on agricultural commodities, mainly cereals, in field as well as during storage. As a defensive response of the host plant, Fusarium toxins can be metabolized by forming modified mycotoxins, often called "masked" mycotoxins. It has been shown that many modified forms are hydrolysed into the parent mycotoxin during digestion. In order to protect consumer health from the risk of exposure to modified and parent forms of Fusarium toxins, the development of rapid, sensitive and reliable methods for their simultaneous determination in cereals is highly demanded. Currently, fluorescence polarization immunoassay (FPIA) is getting the attention as a screening tool in food safety control due to its simplicity, rapidity, cheapness and reliability. The focus of our work is to develop and validate quantitative FPIAs for simultaneous determination of DON and its acetylated (3-acetyl-DON, 15-acetyl-DON) and glycosylated forms (DON-3-glucoside) and T-2/HT-2 toxins and their glycosylated forms (T2-glucoside, HT2-glucoside) in wheat. A fluorescein-label (tracer) of DON (DON-FL) and four T2- and HT2-fluerescein tracers (T2-FL, HT2-FL1a, HT2-FL1b and HT2-FL2) were synthesized and purified. The assessment of the antibody-tracer binding was performed using four DON monoclonal antibodies (MAbs), and ten T2-glucoside MAbs, one HT-2 MAb and two T-2 MAbs, at different concentrations. Concerning the FPIA for the determination of DON, its acetylated and glycosylated forms, the highest antibody-tracer binding was observed for the clone 22/DON-FL combination, while in the FPIA for the determination of T-2/HT-2 toxins and their glycosylated forms, the highest bindings were observed for thirteen T2-glucoside MAbs with T2-FL and HT2-FL1b combinations, as well as for HT-2 MAb/HT2-FL1a combination and for T-2 MAb/T2-FL and HT2-FL1a combinations. Competitive FPIAs were performed with the selected antibody combinations. In particular, FPIA for the determination of DON, its acetylated and glycosylated forms showed IC50=23.5 ng/mL for DON and exhibited 204%, 45% and 11% as cross-reactivity, respectively, for 3-acetyl-DON, DON-3-glucoside and 15-acetyl-DON. While, among the selected combinations for T-2 and HT-2, the HT-2/HT2-FL1a combination exhibited 80% as cross-reactivity for T-2 and its glycosylated form and the highest sensitivity, with IC50 = 2.0, 2.6 and 2.7 ng/mL for HT2, T-2 and T2-glucoside, respectively. These findings showed the applicability of the developed FPIAs to the determination of parent and modified mycotoxins, expressed as sum, in solution. This work was supported by the MYCOKEY project which has received funding from the European Union's Horizon 2020 research and innovation programme under Grant Agreement No 678781.

Ethnic foods are becoming popular worldwide. Nevertheless, foodborne outbreaks and food recalls due to the contamination of these foods with pathogenic agents, toxins, undeclared allergens and hazardous chemical compounds are increasing in recent years together with their growing popularity. In this context, ad hoc prevention and control strategies are needed, to assess and face the emerging microbiological and toxicological risks associated with the consumption of ethnic foods.

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.

This study reports the fungal and bacterial metabolites associated with natural contamination of 38 composite samples of locally processed rice from five Agro-ecological Zones of Nigeria (AEZs). The samples were evaluated for the presence of microbial metabolites with the Liquid Tandem Mass Spectrometry (LC-MS/MS) technique. Among the identified metabolites, 63 fungal and 5 bacterial metabolites were measured at varying concentrations and occurrence levels. Fusarium toxins had the highest incidence of 79% but occurred in low amounts with fumonisin B1 (FB1) having the highest percentage incidence of 39.5% and a mean of 18.52 µg/kg. Among the Aspergillus toxins, aflatoxins (AFs) occurred in 36.9% of the rice samples with aflatoxin B1 (AFB1) having the highest occurrence level of 18.4% and a mean value of 5 µg/kg. About 12 metabolites had incidence levels >50%, including beauvericin (BEA) and tryptophol which had occurrence levels of 100%. Among the emerging toxins under evaluation by international organizations such as the European Food Safety Authority (EFSA) and the Food and Agriculture Organization of the United Nations (FAO), citrinin, sterigmatocystin (STER) and beauvericin were detected with maximum values of 207, 125 and 131 ?g/kg respectively. This paper also reports the first documented evidence of the contamination of Nigerian rice by bacterial and Alternaria metabolites, nivalenol, kojic acid, STER, moniliformin, fusaric acid, fumonisin B3, citrinin, 3-nitropropionic acid, andrastin A, cytochalasins, emodin and physicon.

This study reports the distribution of fungal isolates and fungal metabolites that naturallycontaminate locally processed rice from five agro-ecological zones of Nigeria. The fungal specieswere isolated by the dilution plate technique and identified by appropriate diagnostics, whilemetabolites were determined by a liquid chromatographic tandem mass spectrometric method.Aspergillus and Penicillium species were the predominant isolates found in the rice samples whileFusarium spp. were not isolated. The mean fungal count differed significantly (p < 0.05) across thezones and ranged from 9.98 × 102 cfu g-1 in the Southern Guinea Savannah to 96.97 × 102 cfu g-1in the Derived Savannah. For 16 fungal metabolites, selected from 63 positively identified fungalmetabolites based on their concentration and spread across the zones, an occurrence map wasconstructed. The Northern Guinea Savannah recorded the highest contamination of fungalmetabolites while the Sudan Savannah zone recorded the least.

Finger millet (Eleusine coracana) is a subsistence crop grown in Sub-Saharan Africa and the Indian Sub-continent. Fusarium species occurring on this crop have not been reported. Approximately 13% of the Fusarium isolates recovered from finger millet growing at three different locations in eastern Uganda belong to Fusarium verticillioides, and could produce up to 18,600 µg/g of total fumonisins when cultured under laboratory conditions. These strains are all genetically unique, based on AFLP analyses, and form fertile perithecia when crossed with the standard mating type tester strains for this species. All but one of the strains is female-fertile and mating-type segregates 13:20 Mat-1:Mat-2. Three new sequences of the gene encoding translation elongation factor 1-? were found within the population. These results indicate a potential health risk for infants who consume finger millet gruel as a weaning food, and are consistent with the hypothesis that F. verticillioides originated in Africa and not in the Americas, despite its widespread association with maize grown almost anywhere worldwide.

The Fusarium incarnatum-equiseti species complex (FIESC) includes mycotoxigenicspecies associated with several diseases of cereals and other crops. Althoughthese species are considered as moderately aggressive, they are able to producemultiple mycotoxins, including beauvericin, zearalenone, equisetin,fusarochromanone, butenolide as well as the trichothecenes DAS, MAS, FUS-X,DON, NIV, and scirpentriol. Thus, members of FIESC are potential contributors tomycotoxin contamination of cereals. FIESC includes high levels of crypticspeciation as most species within the complex cannot be distinguished from oneanother by morphological traits. However, a previous DNA-based analysis ofhuman isolates from the US resolved FIESC into 28 phylogenetically distinctlineages, or multilocus sequence types (MLSTs). Here, we investigated thephylogenetic diversity of 69 FIESC isolates recovered from cereals grown in Europeand North America by comparison to the previously described MLSTs. Inphylogenetic analyses of the four housekeeping genes EF-1a, RPB2, CaM andTUB2, 4 isolates were resolved within the F. incarnatum clade of FIESC, and all otherisolates were resolved within the F. equiseti clade. However, 8 isolates wereresolved into a lineage that is distinct from all previously described MLSTs,suggesting that they constitute novel MLST within FIESC. Phylogenomic analysis of12 isolates, representing one novel and 11 previously described and MLSTs, inferreda phylogeny that was consistent with but more highly resolved than the phylogenyinferred from fourgenes. Comparative analysis of the genome sequences revealedvariation in distribution of mycotoxin biosynthetic gene clusters. For example, thetrichothecene cluster is present in all nine genomes, whereas the fusarin andzearalenone clusters are present in only three and four genomes respectively.These data indicate that different FIESC MLSTs vary in their genetic potential toproduce and contaminate cereal crops with different mycotoxins.

The understanding of the yeast population dynamics during spontaneous alcoholic fermentation allows to preserve the microbial biodiversity, to use as indigenous fermentation starters so and to improve the organoleptic and sensory properties of the produced wines. However, it is similarly important to investigate the safety aspects of microbial biodiversity, in particular, on the undesired production of biogenic amines (BAs), low-molecular-weight organic bases produced in wine by the activity of microbial-specific amino acid decarboxylases. This study is the first large-scale investigation on vineyard-associated yeast strains from Apulia (Southern Italy). Eight natural must fermentations were carried out by sampling grape (Vitis vinifera) in the most significant production areas for Negroamaro and Primitivo cultivars: Torchiarolo, Copertino, Cutrofiano and Melissano for the former, Galatina, Torchiarolo, Manduria and Gioia del Colle areas for the latter. Yeasts isolates were identified by PCR ribotyping and bioinformatic analysis of the rRNA Internal region denoted as Transcribed Spacer (ITS). The Saccharomyces cerevisiae strains were further identified and differentiate as strain level by evaluating the polymorphism of their interdelta elements. The results of the molecular analyses revealed the presence of twenty different species belonging to 9 genera. In particular, Hanseniaspora uvarum, Metschnikowia pulcherrima and Aureobasidium pullulans were the dominant strains on the grape surface, whereas M. pulcherrima and H. uvarum were predominant during the early fermentation stage. We identified 692 S. cerevisiae isolates and a number of different strain in each of the 8 fermentations strains, ranging from 26 to 55, The strains were assayed for BAs production, either in synthetic media or grape must. Two Pichia manshurica, an Issatchenkia terricola and a M. pulcherrima strains were capable to produce in wine histamine and cadaverine. The production of BAs in the synthetic medium was dissimilar than that detected in wine, thus enhancing significance to assess the yeast BAs production by an "in grape must" assay, in order to reducethe potential health risk for consumer represented by these spoilage yeasts. To the best of our knowledge, this is the primary study regarding the biodiversity and safety aspects of grape-associated yeast strains in this important wine-producing area of Southern Italy.

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

Lentil samples coming from two different countries, i.e. Italy and Canada, were analysed using untargeted H-1 NMR fingerprinting in combination with chemometrics in order to build models able to classify them according to their geographical origin. For such aim, Soft Independent Modelling of Class Analogy (SIMCA), k-Nearest Neighbor (k-NN), Principal Component Analysis followed by Linear Discriminant Analysis (PCA-LDA) and Partial Least Squares-Discriminant Analysis (PLS-DA) were applied to the NMR data and the results were compared. The best combination of average recognition (100%) and cross-validation prediction abilities (96.7%) was obtained for the PCA-LDA. All the statistical models were validated both by using a test set and by carrying out a Monte Carlo Cross Validation: the obtained performances were found to be satisfying for all the models, with prediction abilities higher than 95% demonstrating the suitability of the developed methods. Finally, the metabolites that mostly contributed to the lentil discrimination were indicated. (C) 2017 Elsevier Ltd. All rights reserved.

Several strains of Fusarium isolated from banana were identified previously as F. verticillioides (Sacc.) Nirenberg but described as unable to produce fumonisin. Here we report biochemical and morphological evidence, as well as multilocus phylogenetic analyses based on elongation factor (EF-1 alpha), calmodulin, beta-tubulin, and the second largest subunit of RNA polymerase II (RPB2) sequences, indicating that these isolates represent a unique lineage in the Gibberella fujikuroi species complex related to but distinct from F. verticillioides. Together with previous results of molecular studies, as well as with results of metabolite analyses, crossing experiments, pathogenicity tests and morphological characterization, these new data indicate that these strains isolated from banana represent a new species, Gibberella musae Van Hove et al. sp. nov. (anamorph: Fusarium musae Van Hove et al. sp. nov.), which is described herein.

In recent years a rising common concern is looking at biodiversity concept with a new sight, attempting to evaluate its economical value, as ground step for supporting measures proposed by national governments and international committees. Although this utilitarian view applied to a complex concept could cause an underestimation of the true potential of biological resources, nowadays a wide spectrum of direct and indirect quantifiable values has been recognized as tightly correlated to biodiversity. Microorganism like fungi play a major role in bio-regulatory systems and could represent an extraordinary source of biodiversity and of new compounds; particular relevance is occupied worldwide by strains belonging to toxigenic genera of Aspergillus, Alternaria, Fusarium, and Penicillium representing a great biodiversity for fungal biology. A critical aspect is the quick deposition and right preservation of "wild" toxigenic fungal strains (TFS) in order to avoid potential loss of their metabolic profile, toxigenicity and pathogenicity . In addition, the biochemical profile should be done using the proper media and incubation conditions and confirmed by HPLC and HRMS methods. This is especially important when new records are being provided, or unexpected mycotoxin production by new fungal species. Then, the importance to deposit of "key" TFS in a non-profit Culture Collection by providing a pure monosporic culture. "Key" strain should be clearly identified on basis of ex-type strain, phylogenetic and biochemical uniqueness, genome sequence-data, strains associated to unique or extreme ambient. It is evident that a biological resources, on which public data has been generated, must be available to research community to check when erroneous results are discovered or when new advanced technologies are available for further study and characterization. In this respect, public service culture collections have been performing this function always by providing optimal environment for long-term maintenance and skilled personnel in identification and managing of fungal strains.Recently, great advance has been done in biodiversity by biochemical and molecular characterization of toxigenic fungi, though data streams from culture collections, research centers, and scientific community are not yet fully integrated with those from biochemical and molecular studies. We wish to stress the importance of using the right approaches in new advance studies on biochemical and molecular characterization of TFS to better understand and preserve the taxonomic diversity and guarantee metabolic properties of the toxigenic fungi community.

Aflatoxin B1 (AFB1) is the most harmful mycotoxin produced mainly by filamentous fungi Aspergillus flavus and A. parasiticus, which can occur as natural contaminants of many agricultural commodities, including maize. Several approaches have been experimented for the removal of aflatoxin from contaminated food and feed, including microbial degradation which, however, has not been so far implemented into practical technologies due to concerns on the quality and safety of the decontaminated materials. The aim of this study was to investigate the capability of the edible and cultivated mushroom Pleurotus eryngii (king oyster mushroom) to degrade AFB1. To this purpouse, nine isolates of P. eryngii were grown on one liquid and one agar medium supplemented with 500 ng/ml of AFB1. After 30 days of growth at 30 ? 1 °C both culture media were processed and analyzed for AFB1 by ultra performance liquid chromatography (UPLC/FLD). In the liquid medium (malt extract broth) all the isolates completely detoxified AFB1. In the solid medium (malt extract-agar supplemented with corn flour and wheat, MEASM) the detoxification ranged from 65 to 84%. In the perspective of developing a technology for bioconversion of aflatoxin-contaminated cereals into added-value feeds, we carried out a pilot study at a laboratory-scale on degradation of AFB1 incorporated in a mushroom cultivation substrate similar to that used in mushroom farms. One strain of P. eryngii (ITEM 13681) was able to bioconvert up to 86% of the AFB1 initially contained in the substrate in 28 days. This value did not change significantly until the maturation of fruit bodies, 42 day post inoculation. The presence of 25% aflatoxin-contaminated maize in the mushroom substrate did not result in a significant reduction of either biological efficiency or yield of the mushroom cultivation. Finally, the analysis of mature fruit bodies did not reveal the presence of detectable amounts of AFB1 or its metabolite aflatoxicol, thus ruling out translocation or "carry-over" of AFB1 through the fungal thallus. These findings make a contribution towards the development of a novel technology for remediation of AFB1-contaminated corn through the exploitation of the degradative capability of P. eryngii.

Cereals represent the major staple food for many people at worldwide level.Among the diseases that affect these crops, the occurrence of Fusarium species isrelated to the highest risk for the consumers since many Fusarium can produce awide range of harmful mycotoxins that can be accumulated in the cereal kernels.In particular, Fusarium Head Blight of wheat and other minor cereals is caused by acomplex of species, each provided of specific mycotoxin profiles. Moreover, themain species can vary in the different geographic areas because they can beinfluenced from the changing environmental conditions. Therefore, a reliableidentification of the most occurring species is important for the correct evaluationof the potential toxicological risk of contaminated kernels. 320 samples of wheatand barley were collected in Austria (2011-2012), Germany (2012) and China(2013) and analyzed for the multi-mycotoxin by liquid chromatography-tandemmass spectrometry (LC-MS/MS) and related toxigenic fungi contamination. Amongthe Fusarium mycotoxins mainly detected in 100 wheat samples from China,enniatins (ENNs), deoxynivalenol (DON), its glucoside DON-3-glucoside (D3G), 3-acetyl- deoxynivalenol (DON), zearalenone (ZEN), nivalenol and, only in 6% ofsamples, fumonisins (FUMs) were identified, with a high number of other mycotoxinsoccurring at low concentrations detected. Also in Germany and Austria, the rangeof mycotoxins detected in wheat and barley was high, being beauvericin (BEA),ENNs, DON, D3G and ZEN the most detected mycotoxins. This wide contaminationby mycotoxins of the samples was also reflected in the wide variability of Fusariumspecies isolated and identified. Fungal strains were first identified based on theirmorphological features and therefore confirmed by sequencing calmodulin andelongation factor 1? genes. In wheat collected in China, F. graminearum sensustricto, F. verticillioides, and species of F. incarnatum/equiseti complex were themost frequently isolated. In Germany and Austria, in both barley and wheat, F.graminearum sensu stricto, F. poae, F. acuminatum and F. tricinctum were themost occurring species. Moreover, a population of strains phylogenetically equallydistant from F. acuminatum and F. tricinctum was also characterized from bothcrops, showing a high level of genetic diversity. However, more genetic analysesare needed to evaluate if this latest population is a new genetic entity to bedescribed within the genus Fusarium.

Spontaneous grapes must fermentations are promoted by the indigenous yeasts, that are able to confer a distinctive style and quality to the produced wine. The spontaneous fermentations of grape must are at first dominated by non-Saccharomyces yeasts and, in a final stage the alcoholic fermentation process is completed by dominant S. cerevisiae strains (Bauer and Pretorius, 2000).The autochthonous yeast strains are associated to a specific vineyard niche habitat and their role in natural fermentation allows the production of wines with particular features in each microclimatic area (Pérez-Coello et al., 1999). However, in order to avoid the unpredictability of must spontaneous fermentation, the winemakers employ commercial dry active yeast culture for wine industrial productions.Increasing interest in the application of locally selected yeasts for fermentation management has been reported (Tristezza et al., 2012). The employment of autochthonous strains of S. cerevisiae as starters seems to be preferable since they are adapted to all the constraints related to a specific wine-production area (Lopes et al., 2007) and are thus capable to dominate more efficiently the indigenous microflora during the fermentation process. Moreover, autochthonous yeast strains can assure the preservation and/or the enhancement of the typical oenological and sensory features which could be considered representative of an oenological region (Rodríguez et al., 2010). In the present study, we developed and applied a strategy to select S. cerevisiae strains from a larger number of yeast isolates. This was achieved adopting a number of key parameters indicative of the strains technological and enological properties. S. cerevisiae population has been isolated from natural fermentations of grape musts, which derive from grapes sampled from the six most representative Negroamaro and Primitivo producing-areas in Apulia (Southern Italy). The yeast populations were identified by molecular assays (AFLP and sequencing) and some selected representative strains were subjected to physiological, oenological and technological characterization.At the end of the selection procedure, that lasted three years, three indigenous S. cerevisiae strains (one for Primitivo and two for Negroamaro), characterized by interesting technological and oenological properties, were selected. The three selected strains were evaluated by both laboratory tests and semi-industrial scale fermentations to confirm their ability to act as autochthonous fermentation starters. An optimized procedure was worked out for the production of starter biomasses, in order to test them in Negroamaro and Primitivo wine production, at an industrial scale, in six different wineries. The employment of autochthonous starter cultures for the industrial production of typical wines in Apulia will be discussed.

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.

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

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.

Oenococcus oeni is the most important LAB species involved in MLF. Highgenotypic heterogeneity of O. oeni strains and a further divergence within thebacterial strain population has been demonstrated. Genotypic diversityseems to be correlated with different metabolic characteristics of bacteria andthis may affect the organoleptic properties of obtained wines. The aim ofthis work was to detect several genes involved in important metabolic pathways(i.e. citrate, sulphur and arginine metabolisms) in 10 indigenous O. oeni strainsselected from Negroamaro wine, a red table wine (Apulia, Italy).This work revealed several new genetic markers that made possible to use a PCRdetection approach to investigate strain heterogeneity for a large range ofgenes encoding enzymes of oenological relevance.

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.

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.

In this study, the effects of pure olive phenolic compounds and olive mill wastewater (OMWW) (after membrane filtration treatments) on Aspergillus flavus growth and aflatoxin B1 (AFB1) production, were investigated. Five OMWWs coming from Greek (Lianolia, Koroneiki and Asprolia) and Italian (Cellina di Nardò and Coratina) olive oil cultivars, opportunely filtered using a membrane system, were added at two concentrations (5 and 15%) to growth medium, in order to evaluate their effect on A. flavus growth and AFB1 production. The OMWW fractions treatment, after 6 days of incubation, did not inhibit the fungal growth rate, but at 15% concentration significantly reduced the AFB1 production (ranging from 88 to 100%). A similar approach was used for caffeic acid, hydroxytyrosol, tyrosol and verbascoside, the major pure phenolic compounds identified in OMWW fractions. They were evaluated at increasing doses (10, 50 and 100 ?g/ml) following both AFB1 production and fungal growth. At the highest concentration (100 ?g/ml) all pure compounds showed a reduction of about 99% of AFB1 production without any influence on fungal growth. This is the first time in which OMWWs and their main phenolics were used in the treatments against AFB1 production. The results obtained could provide possible new strategies for preventing AFB1 food contamination using olive polyphenols and OMWW fractions with anti-aflatoxigenic effect, and permitting to harness in a sustainable way an olive oil by-product.

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

Table olives are one of the most important fermented food in the Mediterranean countries. Apart from lactic acid bacteria and yeasts that mainly conduct the olive fermentation, molds can develop on the brine surface, and can have either deleterious or useful effects on this process. From the food safety point of view, occurring molds could also produce mycotoxins, so, it is important to monitor and control them. In this respect, identification of molds associated to two Italian and two Greek fermented black table olives cultivars, was carried out. Sixty strains were isolated and molecularly identified as Penicillium crustosum (21), P roqueforti (29), P paneum (1), P expansum (6), P. polonicum (2), P commune (1). A group of 20 selected isolates was subjected to technological (beta-glucosidase, cellulolytic, ligninolytic, pectolytic, and xylanolytic activities; proteolytic enzymes) and safety (biogenic amines and secondary metabolites, including mycotoxins) characterization. Combining both technological (presence of desired and absence of undesired enzymatic activities) and safety aspects (no or low production of biogenic amines and regulated mycotoxins), it was possible to select six strains with biotechnological interest. These are putative candidates for future studies as autochthonous co-starters with yeasts and lactic acid bacteria for black table olive production.

Fungal culture collections are important to biologists, microbiologists,epidemiologists and others involved in health and natural sciences. Theimprovement of techniques and methods for fungal isolation and preservation hascontributed to maintain large microbial collections, which represent a rich sourceof biological sciences research, especially taxonomic, pathological and biodiversitystudies as well as industrial applications. The collection centers are responsiblefor repository reference strains and for the maintenance of these microorganisms.The ITEM Microbial Culture Collection of ISPA (Institute of Sciences and of FoodProduction) includes more than 10,000 strains belonging to various agro-food microorganismswith phytopathological and toxicological signi?cance. These microorganismsare mainly fungal pathogens belonging to toxigenic genera of Fusarium, Aspergillus, Alternaria and Penicillium. This collection is a remarkable resource inthe ?ght against mycotoxins: the increasing number of toxigenic fungi included inthis collection ensures an original genetic source for biotechnological applicationsin several ?elds of research, contributing to knowledge improvement about fungalbiology and strategies development for reducing mycotoxin contamination.

In recent years a rising common concern is looking at biodiversity concept with a new sight, attempting to evaluate its economical value, as ground step for supporting measures proposed by national governments and international committees. Although this utilitarian view applied to a complex concept could cause an underestimation of the true potential of biological resources, nowadays a wide spectrum of direct and indirect quantifiable values has been recognized as tightly correlated to biodiversity. Fifty percent of the living biomass on the planet is microbial and microorganisms provide an important source of genetic information for molecular biology and biotechnology. At this respect, the direct-use values is easily perceived and continuously growing thanks to the relevant contribution of biotechnologies, and the possibility to preserve biological resources through long-term conservation of genetic resources. Fungi play a major role in bio-regulatory systems in natural ecosystems and could represent an extraordinary source of new compounds, with a large range of secondary metabolites having biological activities of great ecological relevance, from crop protection to negative impact on humans and domesticated animals.The Agro-Food Microbial Culture Collection "ITEM" (http://server.ispa.cnr.it/ITEM/Collection/), joined to the work for years of researchers in the Institute of Sciences of Food Productions, allows to produce, purify, and characterize novel bioactive metabolites obtained by growing fungal pathogens belonging to several genera. Thousands strains belonging to toxigenic genera of Fusarium, Aspergillus, Alternaria, and Penicillium, represented a great biodiversity in the ITEM collection to deepen the knowledge on fungal biology and strategies development for reducing mycotoxin contamination. Yeast and lactic bacteria strains with peculiar properties has been also preserved and characterized for autochthonous industrial fermentation of typical Apulian wines, table olive and dairy products. Probiotic bacteria are applied for functional foods. A new species of Penicillium from dryed-meat has been isolated and characterized, with possible application for safe seasoning. In general, microorganisms of agro-food interest are preserved and may represent a new frontier of discovery of novel metabolites to be used as safe and environmentally friendly agrochemicals. ITEM take part of the Italian Network of Genetic Resource - BioGenRes (www.biogenres.cnr.it/); and of the European Project on Microbial Resource Research Infrastructure - MIRRI (www.mirri.org/).

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 the last two decades knowledge on lactic acid bacteria (LAB) associated with wine has increased considerably. Investigations on genetic and biochemistry of species involved in malolactic fermentation, such as Oenococcus oeni and of Lactobacillus have enabled a better understand of their role in aroma modification and microbial stability of wine. In particular, the use of molecular techniques has provided evidence on the high diversity at species and strain level, thus improving the knowledge on wine LAB taxonomy and ecology. These tools demonstrated to also be useful to detect strains with potential desirable or undesirable traits for winemaking purposes. At the same time, advances on the enzymatic properties of wine LAB responsible for the development of wine aroma molecules have been undertaken. Interestingly, it has highlighted the high intraspecific variability of enzymatic activities such as glucosidase, esterase, proteases and those related to citrate metabolism within the wine LAB species. This genetic and biochemistry diversity that characterizes wine LAB populations can generate a wide spectrum of wine sensory outcomes. This review examines some of these interesting aspects as a way to elucidate the link between LAB diversity with wine aroma and flavour. In particular, the correlation between inter- and intra-species diversity and bacterial metabolic traits that affect the organoleptic properties of wines is highlighted with emphasis on the importance of enzymatic potential of bacteria for the selection of starter cultures to control MLF and to enhance wine aroma.

There is an urgent need to study in Europe the plant exposure to mycotoxin risk due to the identification of new toxigenic species, the continuous evolution of species profile on the food crops and climate changes that influence the quality of level of toxigenic fungi colonization of plant hosts. In particular, Fusarium and Aspergillus problem in Europe has enormous importance; recent epidemics in wheat in some areas of Northern and Central Europe and in grape in southern Europe have brought this problem into focus again. This concern has driven many efforts at EU level aimed to harmonize strategies for mycotoxinreduction in food and feed chain. This is the target of a large collaborative project of four-year duration (MYCORED as acronym), that was approved within the European FP7- Food, Agriculture, and Biotechnologies Work Programmes (www.mycored.eu). MYCORED aims to develop strategic solutions for reducing mycotoxin contamination in major crops. Novel methodologies, efficient handling procedures and information/dissemination, and educational strategies are considered in a context of multidisciplinary integration of know-how and technology to reduce mycotoxin exposure worldwide. The direct involvement of ICPC countries (Argentina, Egypt, Russia, South Africa) and international organizations (CIMMYT, IITA) together with strong scientific alliances with international experts and national and international societies for mycotoxicology is a strong point of the project through sharing experiences and resources from several past/ongoing mycotoxin projects in a global context. Similarly, the International Society for Mycotoxicology (ISM) (http://www.mycotoxsociety. org) aims at increasing scientific knowledge concerning biology, chemistry and any sciences/disciplines related to mycotoxins and toxigenic fungi, through membership networking, scientific meetings, symposia, discussions, technical courses and publications. In this context, it would be extremely important that MYCORED and ISM develop a network of cooperation-interaction with the whole scientific community in order to contribute to the efforts for harmonizing both research and legislation on mycotoxins.

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.

The diversity of indigenous Oenococcus oeni strains was investigated by molecular and biochemical characterization of isolates from Malvasia Nera wine, an economically important red wine of the Salento Region (Apulia, Italy), during spontaneous malolactic fermentation (MLF). A total of 82 isolates of this species, identified by species-specific PCR and 16S rDNA sequence analysis, were molecularly characterized by the Amplified Fragment Length Polymorphism (AFLP) technique. Three main groups resulted by clustering analysis and showed intraspecific homology higher than 50% and a total of seven subgroups, with similarity values ranged from 80 to 98%, were obtained within these groups. Enzymatic activities, such as esterase, ²-glucosidase, protease, and the consumption rate of L-malic acid, citric acid, acetaldehyde and arginine were assessed in the representative strains according to AFLP analysis. The results displayed different enzymatic activities and consumption rates of tested metabolites among the strains. No correlation between molecular and biochemical data was observed. The evidence of biochemical variability observed among ‘Malvasia Nera’ strains demonstrated that the wine aroma can be modulated depending on the strains involved in the MLF. Hence, the heterogeneity existing within natural O. oeni populations represents an interesting ecological source that can be useful for technological purposes.

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 impact of climate change has been identified as an emerging issue for food security and safety, and the increased incidence of mycotoxin contamination in maize over the last two decades is considered a potential emerging hazard. Disease control by chemical and agronomic approaches is often ineffective and increases the cost of production; for this reason the exploitation of genetic resistance is the most sustainable method for reducing contamination. The review focuses on the significant advances that have been made in the development of transcriptomic, genetic and genomic information for maize, Fusarium verticillioides molds, and their interactions, over recent years. Findings from transcriptomic studies have been used to outline a specific model for the intracellular signaling cascade occurring in maize cells against F. verticillioides infection. Several recognition receptors, such as receptor-like kinases and R genes, are involved in pathogen perception, and trigger down-stream signaling networks mediated by mitogen-associated protein kinases. These signals could be orchestrated primarily by hormones, including salicylic acid, auxin, abscisic acid, ethylene, and jasmonic acid, in association with calcium signaling, targeting multiple transcription factors that in turn promote the down-stream activation of defensive response genes, such as those related to detoxification processes, phenylpropanoid, and oxylipin metabolic pathways. At the genetic and genomic levels, several quantitative trait loci (QTL) and single-nucleotide polymorphism markers for resistance to Fusarium ear rot deriving from QTL mapping and genome-wide association studies are described, indicating the complexity of this polygenic trait. All these findings will contribute to identifying candidate genes for resistance and to applying genomic technologies for selecting resistant maize genotypes and speeding up a strategy of breeding to contrast disease, through plants resistant to mycotoxin-producing pathogens.

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

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

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.

The natural contamination of sorghum and finger millet by toxigenic fungi and associated mycotoxins has been studied. All the tested sorghum and finger millet samples were found to be contaminated by Fusarium and Aspergillus species. Sorghum was considerably more likely to be contaminated by both genera than finger millet. Penicillium, Alternaria, Rhizopus and Epicoccum species were also present in both grains albeit at lower frequencies. Multimycotoxin analysis using LC-MS/MS revealed the contamination of sorghum and finger millet by 84 and 62 metabolites, respectively. The prevalence of major mycotoxins was lower than 15% in sorghum except zearalenone that occurred in one third of the samples at average level of 44 ?g/kg. In finger millet major mycotoxins occurred at a prevalence of 6-52% with zearalenone being the dominant and occurring at average level of 76 ?g/kg. Aflatoxins B1, B2, G1, G2 and M1 were detected in at least one sorghum sample while only aflatoxins B1 and G1 were present in finger millet samples. The average aflatoxins B1 and G1 concentrations in sorghum have been higher than European standards. But the level of B2, G2 and M1 in sorghum and that of B1 and G1 in finger millet have been lower. Apart from aflatoxin precursors and other Fusarium metabolites, a broad range of additional metabolites were detected in sorghum and finger millet.

The MycoKey app is developed as an ICT solution to facilitate mycotoxin risks mitigation by various stakeholders in the chain. Different work packages of MycoKey generate, validate and integrate knowledge that would provide useful information for risk assessment and would help to raise awareness, alert and specifically notify stake holders and provide options for mitigation of mycotoxin risks. This knowledge needs to be customized in order to effectively assist stakeholders. The MycoKey app, a mobile accessible platform, will deliver this customized information on a smartphone, tablet or computer. This app will generate a dashboard experience for accessing all relevant information for growers, advisors, grower associations, stakeholders in the production chain as well as policy-makers. It provides information on the risk of mycotoxins and, when required, will suggest management activities to mitigate and reduce risks. The app is user protected by a personal password and data can be private, shared with friends and advisors or anonymized and shared to other stakeholders. Governmental planners and policy makers will have access to shared, public databases and satellite data, as such biomass indices, land-use and mycotoxin risks can be estimated per region. The MycoKey app has different functionalities for smart phone (data entry and retrieval) and computer platforms (data entry and retrieval and analysis). Recalculation using different intervention strategies allows integration of management strategies in the risk model and calculations of "what if" scenarios. We hope to demonstrate the MycoKey app in Ghent for the first time!

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.

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.

The potential risk of exposure to fumonisin B2 (FB2) in the grape-wine chain has recently been revealed after a report of Aspergillus niger in grapes and its ability to produce FB2 and FB4. The occurrence of these two fumonisins in wine was investigated by LC/MS/MS in 51 market samples (45 red, five white and one rose wine) produced in various Italian regions. Nine samples of red wine were found to be contaminated by fumonisin B2 at levels ranging from 0.4 to 2.4 ng/ml, while FB4 was not detected in any of the tested samples. This is the first report on the natural occurrence of FB2 in wine, indicating that, although at low levels, there is a potential risk of FB2 exposure for the wine-consumer.

Oenococcus oeni is the main lactic acid bacterium involved in Malolactic Fermentation since its high adaptation capacity in wine (1). Extensive studies, carried out over the years, furnished a considerable amount of information on the genetic, physiology and metabolism of this bacterium (2-4). The increasing acquisition of data about the secondary metabolic activities exhibited by O. oeni, which impact strongly to sensory properties of wine, stimulated the investigations on variability within indigenous populations isolated from various winemaking environments.Indeed, strains can modified differently the flavour, quality and safety of wine according to their to metabolic diversity and, due to the economic importance of wine, great interest has been addressed to the study of intraspecific heterogeneity of O. oeni (5, 6).In this work, the detection of several genes involved on important metabolic pathways (i.e. citrate, sulphur and arginine metabolism) was performed on 10 indigenous O. oeni strains of Negroamaro wine, a table red wine (Apulia, Italy). These strains were selected from 95 isolates, collected during a spontaneous malolactic fermentation, according to the results of Amplified Fragment Length Polymorphism (AFLP) analysis. It was screened a total of 16 genes, most of them (11) never assayed before on O. oeni. All strains possessed 10 genes encoding enzymes such as malolactic enzyme (mleA), esterase (estA), citrate lyase ?citD, citE and citF), citrate transporter (maeP), ?-acetolactate decarboxylase (alsD), ?-?acetolactatesynthase (alsS), S-adenosylmethionine synthase (metK) and cystathionine ?-lyase (metC) and resulted negative in the detection of genes encoding cystathionine ?-lyase (metB), ornithine transcarbamylase (arcB) and carbamate kinase (arcC) (table 1.). The sequence of PCR fragments of 11 genes of a representative strain (ITEM 15929) were compared to those of three reference O. oeni strain. The indigenous strain phylogenetically resulted more similar to PSU-1 and ATCC BAA1163 than AWRI B429. The present study provides information on population structure of the species and describes new genetic markers useful for detecting the genetic potential of O. oeni strains to contribute to aroma production and to improve the quality of wine.Table 1. Results of PCR detection of different enzyme-encoding genes in a population of 10 O.oeni strains.Target geneAccession number protein activities

Table olives are one of the most important traditional fermented vegetables in Europe and their world consumption is increasing. In the Greek system, table olives are produced by natural fermentation process, that is not predictable and strongly influenced by the physical-chemical conditions and by the presence of microorganisms contaminating the olives, In this study , we have developed and validated a novel procedure for table olive production based on the use of a mixed yeast/bacteria starter.

Table olives represent one important fermented product in Europe and, in the world, their demand is constantly increasing. At the present time, no systems are available to control black table olives spontaneous fermentation by the Greek method. During this study, a new protocol for the production of black table olives belonging to two Italian (Cellina di Nardò and Leccino) and two Greek (Kalamàta and Conservolea) cultivars has been developed: for each table olive cultivar, starter-driven fermentations were performed inoculating, firstly, one selected autochthonous yeast starter and, subsequently, one selected autochthonous LAB starter. All starters formulation were able to dominate fermentation process. The olive fermentation was monitored using specific chemical descriptors able to identify a first stage (30 days) mainly characterized by aldehydes; a second period (60 days) mainly characterized by higher alcohols, styrene and terpenes; a third fermentation stage represented by acetate esters, esters and acids. A significant decrease of fermentation time (from 8 to 12 months to a maximum of 3 months) and an significant improvement in organoleptic characteristics of the final product were obtained. This study, for the first time, describes the employment of selected autochthonous microbial resources optimized to mimic the microbial evolution already recorded during spontaneous fermentations.

Two approaches were developed in order to select microorganisms suitable to be used in olive millwastewaters bioremediation. By the first approach, three hundred yeasts were isolated from fiveindustrial mills and identified by molecular analysis. The different strains were selected accordingto their capacity to grow in OMW (olive mill wastewaters) as the sole carbon source and to reducephenolics, chemical oxygen demand (COD) and antimicrobial compounds. One Geotrichum candidumisolate was used to set up a whole-cell immobilization system in calcium alginate gel andthe COD and phenolic reduction obtained using the immobilized cells showed respectively a 2.2-and 2-fold increase compared to the removal obtained using free cells. By the second approach, anew protocol was developed to isolate and select aerobic microorganisms from different industrialsamples and environmental niches (soils, OMWs) and able to detoxify olive mill wastewaters.

Egypt is the most important producer and consumer Country of table olives in the world. Within MARSADEV Project, a survey on table olives commercial products collected in the Northwestern region of Matrouh Governorate in Egypt was performed. Low safety conditions were observed in these products. Some of them are affected by the occurrence of high counts of Pseudomonas, Staphylococci and Enterobacteriaceae and some of them by the presence of biogenic amines. All tested samples resulted very poor in compounds related to microbial activities, such as organic acids and olives-derived phenols. Also, from the organoleptic point of view, odour profiles of tested olives are very modest. Scarce hygienic conditions were registered within domestic or small industrial plants along the whole production chain: from the harvest, to the processing and to the table olives packaging and storage. Several operations, such as the use of microbial starters, the monitoring of the fermentation process, together with some operational precautions, i.e. the heat treatment of water before olive soaking and the use of gloves during all operations, have been proposed to improve the process safety. The use of selected yeasts as starter for table olives production and the control of physical-chemical parameters during fermentation allowed to control the process, to sensitively improve organoleptic and safety traits of table olives, to reduce time required to end the transformation process from 8 to 2 months.Guidelines suggesting good practices and describing the most important operational steps to be followed for table olives processing have been produced for Bedouin rural communities living in this area, in particular for women within the concerned communities.

Non-destructive evaluation of vegetables by Computer Vision Systems (CVSs) makes possible to check their quality level in an objective and consistent way along the whole supply chain up to the final users. CVSs have been proven to be successful when applied to unpackaged products.The proposed approach aimed to enable this analysis on packaged fresh-cut lettuce with minimum constraints on the acquisition phase and without any care to flatten the surface of the bag facing the camera. A deep-learning architecture, based on Convolutional Neural Networks (CNNs), was used to identify regions of the image where the vegetable was visible with minimum colour distortions due to packaging. To meaningfully assess the performance of the system, each lettuce's sample was acquired both through packaging material and without packaging material. The image analysis was applied to both the resulting images to automatically grade their quality level. The results showed that the performance loss due to the presence of packaging is negligible (83% instead of 86%) and that the proposed system can be used to monitor the quality level of fresh-cut lettuce regardless of packaging at all the critical check points along the supply chain.

Two approaches were developed in order to select microorganisms suitable to be used in olive mill wastewaters bioremediation. By the first approach, three hundred yeasts were isolated from five industrial mills and identified by molecular analysis. The different strains were selected according to their capacity to grow in OMW (olive mill wastewaters) as the sole carbon source and to reduce phenolics, chemical oxygen demand (COD) and antimicrobial compounds. One Geotrichum candidum isolate was used to set up a whole-cell immobilization system in calcium alginate gel and the COD and phenolic reduction obtained using the immobilized cells showed respectively a 2.2- and 2-fold increase compared to the removal obtained using free cells. By the second approach, a new protocol was developed to isolate and select aerobic microorganisms from different industrial samples and environmental niches (soils, OMWs) and able to detoxify olive mill wastewaters.

Fusarium Head Blight (FHB) represents one of the most economically worldwidedevasting disease of of durum wheat, causing significant reduction of grain yieldand quality. FHB of wheat is caused by a complex of species belonging mostly toFusarium genus. Many of these species can produce a wide range of mycotoxinsthat can be accumulated in wheat kernels at maturity, among which thetrichotecene, strong protein inhibitors, are the most common. Moreover, eachspecies of Fusarium involved in the FHB is provided of its own specific profile. Thespecies can vary in the different geographical areas because they can beinfluenced from the changing environmental conditions. One-hundred-foursamples of durum wheat were collected in Italy in 2013 and 2014 and analyzed forthe occurrence of trichothecenes by Ultra-Performance LiquidChromatography/Photodiode-Array Detector and zearalenone (ZEA) by highperformanceliquid chromatography with fluorescence detection. The Fusariumspecies isolated from the kernels were first identified based on their morphologicalfeatures and therefore confirmed by sequencing calmodulin and elongationfactor 1? genes. The Fusarium mycotoxin detection varied in 2013 compared to2014 and also according with geographical areas. Deoxynivalenol (DON) wasdetected at a relevant levels only in the samples collected in Central andNorthern Italy, with higher concentrations and incidence in 2014 compared 2013.On the other hand, the T-2 and HT-2 toxins and ZEA occurred at higher levels insamples collected in Southern Italy than in Central Italy and Northern Italy, and in2014 the level of contamination was higher than in 2013. These latter data are alsoreason of the highest concern since 18 out of 20 wheat samples in both 2013 and2014 (range, 100-335 and 155-486 ppb, respectively) were over the recommendedlimits suggested by the European Union for the sum of T-2 and HT-2 toxins in thewheat kernels. The mycotoxin contamination that occurred in the kernels was alsoreflected in the spectrum of Fusarium species isolated and identified. Fusariumgraminearum sensu stricto was the most occurring species when the DONoccurred at high levels and F. langsethiae was the species isolated frequentlywhen T-2 and HT-2 toxins were detected. These data showed that a real mycotoxinrisk related to Fusarium mycotoxins does exist along the whole Italy, but they varyaccording with the geographical areas and year of sampling.

Twenty-four samples including 14 functional foods and 10 spices obtained from Chinese markets were examined for their mould profile. The mycotoxin contamination levels were also determined by an optimized HPLC-FLD method. 124 fungal isolates belonging to four different genera were recovered with Aspergillus and Penicillium as predominant fungi, with an incidence of 66.1% and 15.3%, respectively. In functional foods Aspergillus niger section (57.1%) was isolated more frequently, followed by Aspergillus flavi section (50.0%) and Aspergillus ochraceus section (21.4%), with the most contaminated samples being Coix seeds. Similar fungal presence and frequency were encountered in spice with A. niger section group (60.0%) and A. flavi section (40.0%) as main fungi. Cumin and Pricklyash peel samples showed the highest fungal contamination. Four functional foods and three spices were found to be positive at low levels for mycotoxins including aflatoxin B1 (up to 0.26 ?g/kg) and ochratoxin A (OTA) (5.0 ?g/kg). The more frequently detected mycotoxin was AFB1 (16.7%).

Grape berries attacked by Lobesia botrana larvae are more easily infected by Aspergillus section Nigri (black aspergilli) ochratoxigenic species. Two-year field trials were carried out in Apulia (Italy) to evaluate a bioinsecticide control strategy against L. botrana and the indirect effect on reducing ochratoxin A (OTA) contamination in vineyards. A commercial Bacillus thuringiensis formulate and an experimental Beauveria bassiana (ITEM-1559) formulate were tested in two vineyards cultivated with the same grape variety, Negroamaro, but with two different training systems (espalier and little-arbor techniques). In both years and training systems the treatments by B. bassiana ITEM-1559 significantly controlled L. botrana larvae attacks with effectiveness similar to B. thuringensis (more than 20%). A significant reduction of OTA concentrations (up to 80% compared to untreated controls) was observed only in the first year in both training systems, when the metereological parameters prior to harvest were more favorable to the insect attack. Results of field trials showed that B. bassiana ITEM-1559 is a valid bioinsecticide against L. botrana and that grape moth biocontrol is a strategy to reduce OTA contamination in vineyard in seasons with heavy natural infestation.

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarittm into nine or more genera, and remove important taxa such as those in the E solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within alid between research communities, and at the same time support strong scientific principles and good taxonomic practice.

Species of Aspergillus section Nigri are commonly associated with maize kernels, and some strains can produce fumonisin mycotoxins. However, there is little information about the extent to which these fungi contribute to fumonisin contamination in grain, the damage they cause to maize ears, or their effects on maize seed germination and seedling health. We compared fumonisin-producing and nonproducing strains of A. niger, A. welwitschiae, A. phoenicis, A. tubingensis, and A. carbonarius from the United States and Italy in laboratory and field studies to assess their ability to contribute to fumonisin contamination, to cause maize ear rot, and to affect seed germination and seedling growth. In laboratory experiments, some strains of each Aspergillus species reduced germination or seedling growth, but there was high variability among strains within species. There were no consistent differences between fumonisin-producing and nonproducing strains. In field studies in Iowa and Illinois, strains were variable in their ability to cause ear rot symptoms, but this was independent of the ability of the Aspergillus strains to produce fumonisins. Contamination of grain with fumonisins was not consistently increased by inoculation with Aspergillus strains compared with the control, and was much greater in F. verticillioides-inoculated treatments than in Aspergillus-inoculated treatments. However, the ratio of the FB analogs FB2 and FB1 was altered by inoculation with some Aspergillus strains, indicating that FB2 production by Aspergillus strains occurred in the field. These results demonstrate the pathogenic capabilities of strains of Aspergillus in section Nigri, but suggest that their effects on maize ears and seedlings are not related to their ability to produce fumonisins, and that fumonisin contamination of grain caused by Aspergillus spp. is not as significant as that caused by Fusarium spp.

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.

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.

Table olives are one of the most important traditional fermented vegetables in Europe and their world consumption is constantly increasing. In the Greek style, table olives are obtained by spontaneous fermentations, without any chemical debittering treatment. Evolution of sugars, organic acids, alcohols, mono and polyphenol compounds and volatile compounds associated with the fermentative metabolism of yeasts and bacteria throughout the natural fermentation process of the two Italian olive cultivars Cellina di Nardò and Leccino were determined. A new protocol was developed and applied aimed at the technological characterization of LAB and yeast strains as possible candidate autochthonous starters for table olive fermentation from Cellina di Nardò and Leccino cultivars. The study of the main physical, chemical and aromatic parameters during fermentation helped to determine chemical descriptors that may be suitable for monitoring olive fermentation. In both the analyzed table olive cultivars, aldehydes proved to be closely related to the first stage of fermentation (30 days), while higher alcohols (2-methyl-1-propanol; 3-methyl-1-butanol), styrene, and o-cymene were associated with the middle stage of fermentation (90 days) and acetate esters and acetic acid with the final step of olive fermentation (180 days).

Table olives are one of the most important traditional fermented vegetables in Europe and their worldconsumption is constantly increasing. Conservolea and Kalam
ata are the most important table olivesGreek varieties. In the Greek system, the final product is obtained by spontaneous fermentations,without any chemical debittering treatment. This natural fermentation process is not predictable andstrongly influenced by the physical-chemical conditions and by the presence of microorganismscontaminating the olives. Natural fermentations of Conservolea and Kalam
ata cultivars black olives were studied in order to determine microbiological, biochemical and chemical evolution during the process. Following the process conditions generally used by producers, in both cultivars, yeasts were detected throughout the fermentation, whereas lactic acid bacteria (LAB) appeared in the last staged of the process. A new optimized specific protocol was developed to select autochthonous yeast and LAB isolates that can be good candidates as starters. These microorganisms were pre-selected for their ability to adapt to model brines, to have beta-glucosidase activity, not to produce biogenic amines. Chemical compounds deriving by microbiological activities and associated to the three different phases (30, 90 and 180 days) of the fermentation process were identified and were proposed as chemical descriptors to follow the fermentation progress.

Filamentous fungi are the main pathogens of withered grapes destined for passito wine production. Knowledge of which species inhabit these post-harvest fruits and their pathogenicity is essential in order to develop strategies to control infection, but is still scarce. This study investigated the predominant mycobiota of withered grapes through a cultivation-dependent approach. Strain and species heterogeneity was evidenced on examining isolates collected over three consecutive years. Colony morphology and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis revealed the occurrence of several phenotypes and haplotypes, respectively. Strains were phylogenetically analyzed based on sequence typing of different genes or regions (e.g. calmodulin, ?-tubulin and internal transcribed spacer region). Beside the most common necrotrophic-saprophytic species of Penicillium, Aspergillus, Alternaria and Botrytis species responsible for fruit rot, other saprobic species were identified (e.g. Trichoderma atroviride, Sarocladium terricola, Arthrinium arundinis and Diaporthe eres) generally not associated with post-harvest fruit diseases. Species such as Penicillium ubiquetum, Cladosporium pseudocladosporioides, Lichtheimia ramosa, Sarocladium terricola, Diaporthe nobilis, Bipolaris secalis, Paraconiothyrium fuckelii and Galactomyces reessii that had never previously been isolated from grapevine or grape were also identified. Moreover, it was not possible to assign a species to some isolates, while some members of Didymosphaeriaceae and Didymellaceae remained unclassified even at genus level. This study provides insights into the diversity of the epiphytic fungi inhabiting withered grapes and evidences the importance of their identification to understand the causes of fruit diseases. Finally, phylogenetic species delimitation furnished data of interest to fungal taxonomy.

The aim of this work was to recover and identify the phenolic compounds from olive mill waste water (OMWW) samples belonging to two Italian (Cellina and Coratina) and three Greek (Asprolia, Lianolia and Koroneiki) olive cultivars. The OMWWs were processed using membrane technologiesto obtain three fractions: microfiltrate (MF), ultrafiltrate (UF) and nanofiltrate (NF). These steps allowed to purify the OMWWs in order to achieve fractions with different profile and concentrations of polyphenols. In particular, the amount of polyphenols ranged from 2456 ?g/mL to 5284 ?g/mL in MF; from 1404 ?g/mL to 3065 ?g/mL in UF and from 373 ?g/mL to 1583 ?g/mL in NF. Among the cultivars analyzed Coratina followed by Lianolia showed the highest amount of verbascoside (VB) (308?g /mL in Coratina versus 145?g/mL in Lianolia, respectively) in UF fractions. Furthermore, UF fractions that showed adequate purification degree and polyphenol enrichments, were used for the identification of the phenolic compounds by liquid chromatography/diode array detection/electrospray ion trap tandem mass spectrometry (LC/DAD/ESI-MSn) analysis. Twenty three compounds, belonging to the following classes of constituents: secoiridoids and their derivatives, phenyl alcohols phenolic acid and derivatives, and flavonoids, were identified in almost all the UF fractions of the different cultivars. Finally, differences were observed among the cultivars regarding the presence of elenolic acid derivatives, hydroxytyrosol glucoside, and ?-hydroxyverbascoside diasteroisomers. The results obtained showed that OMWW can be considered as raw material for the isolation of valuable bioactive compounds able to be used in food, cosmetic and pharmaceutical industry.

Aflatoxins are highly toxic carcinogens that contaminate crops worldwide. Previous studies conducted in Nigeria and Ghana found high concentrations of aflatoxins in pre- and post-harvest maize. However, little information is available on the population structure of Aspergillus Sect. Flavi in West Africa. We determined the incidence of Aspergillus Sect. Flavi and the level of aflatoxin contamination in 91 maize samples from farms and markets in Nigeria and Ghana. Aspergillus spp. were recovered from 61/91 maize samples and aflatoxins B-1 and/or B-2 occurred in 36/91 samples. Three samples from the farms also contained aflatoxin G(1) and/or G(2). Farm samples were more highly contaminated than were samples from the market, in terms of both the percentage of the samples contaminated and the level of mycotoxin contamination. One-hundred-and-thirty-five strains representative of the 1163 strains collected were identified by using a multilocus sequence analysis of portions of the genes encoding calmodulin, beta-tubulin and actin, and evaluated for aflatoxin production. Of the 135 strains, there were 110 - Aspergillus flavus, 20 - Aspergillus tamarii, 2 - Aspergillus wentii, 2 - Aspergillus flavofurcatus, and 1 - Aspergillus parvisclerotigenus. Twenty-five of the A. flavus strains and the A. parvisclerotigenus strain were the only strains that produced aflatoxins. The higher contamination of the farm than the market samples suggests that the aflatoxin exposure of rural farmers is even higher than previously estimated based on reported contamination of market samples. The relative infrequency of the A. flavus S-BG strains, producing small sclerotia and high levels of both aflatoxins (B and G), suggests that long-term chronic exposure to this mycotoxin are a much higher health risk in West Africa than is the acute toxicity due to very highly contaminated maize in east Africa. (C) 2014 Elsevier Ltd. All rights reserved.

Polyphenols, secondary metabolites widely present in plant kingdom, are known for their positive effects on human health, such as treatments of degenerative disease and cancer. Many dietary polyphenols show anti-inflammatory, immunomodulatory and anti-oxidant properties and they are proposed as chemopreventive agents for many skin disorders and cancer. Exposure to solar UV radiation is widely considered to cause skin cancer and a consistent carcinogenic dose derives from UVA, causing several skin disorders as consequence of free radicals generation and DNA damages. Polyphenols, secondary metabolites widely present in plant kingdom, are known for their positive effects on human health, such as treatments of degenerative disease and cancer. Many dietary polyphenols show anti-inflammatory, immunomodulatory and anti-oxidant properties and they are proposed as chemopreventive agents for many skin disorders and cancer. Exposure to solar UV radiation is widely considered to cause skin cancer and a consistent carcinogenic dose derives from UVA, causing several skin disorders as consequence of free radicals generation and DNA damages. In this study, verbascoside, isoverbascoside and tyrosol were investigated for their effect on HEKa (Human Epidermal Keratinocytes adult) cell cultures challenged from UVA-rays. Non-toxic doses of each polyphenol were assayed on HEKa before, during and after the exposure to a damaging dose of UVA: the treatment on HEKa UVA-irradiated caused a decrease of cell viability. Treatment with polyphenols before and after the UVA-irradiation exerted a pro-oxidant effect, while the simultaneous treatment caused a weak decrease of ROS production. The increasing of ROS levels was associated with a pro-apoptotic effect on HEKa, detected by AnnexinV/Propidiun Iodide, mainly evident in surviving cells treated with the polyphenols after the UVA-irradiation. The pro-apoptotic effect was confirmed by the immunodetection of significant changes in the Bax and Bcl-xL protein levels, leading to apoptotic events. The hypothesis that these polyphenols could trigger the apoptosis pathway mainly in UVA-damaged cells, via ROS increase, is here proposed as action mechanism behind their protective effect.

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.

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

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.

Table grapes (cv. Italia) were stored at 5 °C in air or in modified atmosphere with low O2 (1%) and low(0.03-10%) or high (> 20%) CO2. Volatile organic compounds (VOCs) associated with quality (respiration rate,acetaldehyde and ethanol, water status of rachis) and sensory parameters (visual quality, rachis browning,characteristic odour and flavour, off-odour and off-flavour) were evaluated. Low CO2 preserved the quality andsensory parameters, whereas high CO2 caused a fermentative metabolism. A total of 126 VOCs were identified byHS-SPME/GC-MS analysis. Principal component analysis (PCA) identified (Z)-2-hexenal and (E,Z)-2,4-hexadienalas potential marker of freshness; whereas, acetaldehyde, ethanol, 1-butanol, 3-methyl-1-butanol, 1-octanoland 1-nonanol were correlated to fermentation processes. Partial least-square regression analysis (PLSR)was used to identify (R2 =0.95) the main predictors of off-odour as ethanol and ethyl acetate (+) and hexanaland ?-linalool (-).

Fusarium verticillioides is the prevailing species in maize fields producing ear rot and fumonisins that are suspected to be carcinogenic. In this study we analyze the host response in early and late stages after F. verticillioides infection in susceptible and resistant maize kernels sampled in the area neighbouring the point of inoculation. The fungal growth, assayed by qRT-PCR quantification of the tub 2 gene, was eight times lower, in average, in resistant kernels than that detected in susceptible line over the time course of 96 h. During the very early stages of infection (12-24 h after infection) a small proportion of the host transcripts was induced. The number of differentially regulated genes reached 147 at 48 h after infection and decreased to 140 and 98 at 72 and 96 h post infection, respectively. About 1.0% of the genes assayed were differentially expressed and 7.1% of them was assigned to the category cell rescue, defense and virulence. Pathogenesis-related protein-5, endo-1,3-1,4-beta-D-glucanase PRm6, chitinase PRm3 and MYB-like DNA binding protein had a higher level of expression in the resistant line compared to the susceptible one. Since having analyzed the area around the point of infection, the resistant line may activate more efficiently a battery of defense genes, before the invasion by the fungus in non damaged tissues or in adjacent ones. These outcomes indicate that the resistant kernels activated a basal defense programme where the expression of defense genes is controlled by both the host genotype and induced by the pathogen.

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

Table olives are one of the most important traditional fermented vegetables in Southern European countries and their consumption is constantly increasing throughout the world. Today, the industrial production of black table olives is carried out by spontaneous fermentation processes which are not predictable and are strongly influenced by the autochthonous microflora, the physical-chemical conditions, the availability of fermentable substrates and salt content. Evolution of sugars, organic acids, alcohols, mono and polyphenol compounds and volatile compounds associated with the fermentative metabolism of yeasts and bacteria throughout the natural fermentation process of the two Italian olive cultivars Cellina di Nardò and Leccino were determined. A new protocol was developed and applied aimed at the selection of LAB and yeast strains as candidate autochthonous starters for table olive fermentation from Cellina di Nardò and Leccino cultivars.

Fusarium head blight (FHB) is an important disease of wheat worldwide caused mainly by Fusarium graminearum (syn. Gibberella zeae). This fungus can be highly aggressive and can produce several mycotoxins such as deoxynivalenol (DON), a well known harmful metabolite for humans, animals, and plants. The fungus can survive overwinter on wheat residues and on the soil, and can usually attack the wheat plant at their point of flowering, being able to infect the heads and to contaminate the kernels at the maturity. Contaminated kernels can be sometimes used as seeds for the cultivation of the following year. Poor knowledge on the ability of the strains of F. graminearum occurring on wheat seeds to be transmitted to the plant and to contribute to the final DON contamination of kernels is available. Therefore, this study had the goals of evaluating: (a) the capability of F. graminearum causing FHB of wheat to be transmitted from the seeds or soil to the kernels at maturity and the progress of the fungus within the plant at different growth stages; (b) the levels of DON contamination in both plant tissues and kernels. The study has been carried out for two years in a climatic chamber. The F. gramineraum strain selected for the inoculation was followed within the plant by using Vegetative Compatibility technique, and quantified by Real-Time PCR. Chemical analyses of DON were carried out by using immunoaffinity cleanup and HPLC/UV/DAD. The study showed that F. graminearum originated from seeds or soil can grow systemically in the plant tissues, with the exception of kernels and heads. There seems to be a barrier that inhibits the colonization of the heads by the fungus. High levels of DON and F. graminearum were found in crowns, stems, and straw, whereas low levels of DON and no detectable levels of F. graminearum were found in both heads and kernels. Finally, in all parts of the plant (heads, crowns, and stems at milk and vitreous ripening stages, and straw at vitreous ripening), also the accumulation of significant quantities of DON-3-glucoside (DON-3G), a product of DON glycosylation, was detected, with decreasing levels in straw, crown, stems and kernels. The presence of DON and DON-3G in heads and kernels without the occurrence of F. graminearum may be explained by their water solubility that could facilitate their translocation from stem to heads and kernels. The presence of DON-3G at levels 23 times higher than DON in the heads at milk stage without the occurrence of F. graminearum may indicate that an active glycosylation of DON also occurs in the head tissues. Finally, the high levels of DON accumulated in straws are worrisome since they represent additional sources of mycotoxin for livestock. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

Olive oil production generates huge quantities of waste in the form of olive mill waste waters (OMWW), which may have great environmental impact because of their high phytotoxicity, toxicity against aquatic organisms and possible suppression of soil microorganisms. However, this by-product is characterized by a high organic component including numerous interesting compounds, such as polyphenols with valuable biologic properties. Many studies were carried out in order to recover polyphenols by different strategies or to use the aqueous extract of OMWW for the preparation of high value added products.The objective of this work in the within of the EU project BIO-OLEA "Utilization of biophenols from Olea Europea products - Olives, virgin olive oil and olive mill wastewater" was to obtain scientific evidences about the biological activity of several compounds present in OMWW, their action mechanisms and putative utilizations. Polyphenols usually present in OMWW were considered and the effect of each compound was valuated on human keratinocyte cell culture exposed with different timing, to ultraviolet -A rays (UVA) that are known as one of the most important etiological factors in the development of skin disorders such as immunosuppression, photo-aging and photo-carcinogenesis, and cancer. This activity was aimed to find out the possible uses of OMWW based products by defining the biological activity of specific polyphenols present in OMWW.Understanding basic action mechanisms enables us to supply the general knowledge on the biological properties of polyphenols present in the OMWW matrix and to contribute to the technological information useful to enable the producers to manage the OMWW. OMWW represent a rich source of phenol compound with high antioxidant activity and valuable other biological properties. Here it is proposed that some of these polyphenols have the ability to trigger the apoptosis pathways mainly in UVA-damaged cells, via ROS increase, as an action mechanisms behind their protective effect.The implementation of olive oil production by means of different technologies aimed to preserve the OMWW quality and safety could allow the integration of the recovery and utilization of this by-product, in the olive oil production process.

The case study focuses on the role that production and consumption of "traditional vegetables" play in the vegetables food-system in Puglia (Southern Italy).Puglia, leading region for intensive horticulture, boasts an enormous heritage of local vegetable varieties, whose cultivation is still widespread and well established in response to demands of local, international and high gastronomy markets, based on their higher quality and the socio-cultural implications associated. However, a significant part of this agro-biodiversity has been lost in the last fifty years, and the threat of continuing to lose it in the future is serious.The traditional vegetables complement the production and the supply of conventional ones, in a much-diversified scenario of production and consumption styles, representing an example of agro-biodiversity protection and cultivation practices calibrated on the characteristics of Mediterranean environments. The policy makers are investing resources on the protection and enhancement of agro-biodiversity, involving research institutions and promoting actions addressed to seed-saver farmers and consumers.The commercial value of these products is supporting the establishment of forms of association between producers, favoring the creation of brands and facilitating the consumption of local products.

Table olives are one of the most important traditional fermented vegetables in Southern European (Italy, Greece and Spain) countries. In the Greek-style production system, the fruits are placed directly into the brine, thus allowing the natural fermentation to take place. The spontaneous fermentations, that can last 8-12 months, are driven by mixed populations of microorganisms, mainly the epiphytic microbial population of yeasts and lactic acid bacteria (LAB) (Romero et al., 2004). At present, the industrial table olive process is not predictable and depends on the empirical experience of the producers. In order to avoid the unpredictability of the olive spontaneous fermentation, to improve the productive process and to constantly produce high-quality final products, the use of strains of LAB as starter cultures for olive production has been proposed (Sabatini and Marsilioet al., 2008; Panagou et al., 2008; Blana et al., 2014). However, in the last years, the importance and the potential applications of yeasts as starters for table olive processing has been recognized (Arroyo-López et al., 2008, 2012; Bevilacqua et al., 2012). Objectives: In the present work, we have studied the main physical, chemical and aromatic parameters of natural fermentations of Cellina di Nardò, Leccino, Kalamata and Conservolea table olives in order to determine chemical descriptors correlated to microbiological activities and the dynamics of microorganisms in order to select LAB and yeast strains as candidate autochthonous starter cultures. Conclusions: The identified chemical descriptors can be suitable to follow the trend and to control the outcome of the fermentation and a new protocol aimed to the selection of LAB and yeast strains as candidates autochthonous starters has been developed and applied (Bleve et al. 2014 a, b). Selected microbial starters have been successfully used to ferment olives in pilot and industrial-scale and a new method for table olive production has been set up (Bleve et al. 2103). The use of selected autochthonous starter cultures produced fermented table olives with improved organoleptic, sensorial and nutritional characteristics.

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

Figs are an economically important crop in the Mediterranean area. Fungal infection can be observed on figs on the tree, after shriveling, after falling to the ground, and during the drying process. Fungal growth and subsequent mycotoxin production are influenced by a variety of complex interactions between instrinsic and extrinsic factors as well as stress factors and physical damage. The dominant fungal flora in dried figs consisted of Aspergillus section Nigri, Fusarium spp., Aspergillus section Flavi and Penicillium spp. Fungal infection can result in mycotoxin contamination including aflatoxins, citrinin, cyclopiazonic acid, fumonisins, patulin and ochratoxin A. This review describes the major fungal infection and mycotoxin contamination in dried figs.

Lentil (Lens culinaris Medik.) is the fourth most important pulse crop in the world after bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), and chickpea (Cicer arietinum L.). Canada is the world's largest exporter of lentils, while in Italy lentils are a minor legume and can be found in restricted areas. However, Italian lentils present unique and characteristic qualities giving them a higher value, so that many of them have obtained international and national marks linked to their geographical origins, such as "protected geographical indication" (PGI), "traditional food products" (PAT) and Slow Food Presidium. For these reasons, there is a growing demand for analytical methods able to certify the declared geographical origin of lentils, in order to protect consumers and producers from fraud and unfair competition. In the present work, the potential of infrared spectroscopic fingerprinting technique for the geographical origin traceability of lentils was investigated. In particular, lentil samples from two different countries, i.e. Italy and Canada, were collected from retail markets and analysed by Fourier transform near- and mid-infrared spectroscopy (FT-NIR, FT-MIR). After a suitable pretreatment of the raw spectral data, Linear Discriminant Analysis (LDA) was used examining the FT-NIR and FT-MIR fingerprints separately and in combination in order to evaluate the spectral range mostly influenced by geographical origin. The LDA classification results were expressed in terms of recognition and prediction abilities (cross validation and external validation). Good classification results were obtained for both FT-NIR and FT-MIR ranges with FT-MIR one giving better prediction abilities, i.e. 95% and 92% for cross and external validation, respectively. The combination of the FT-MIR and F-NIR did not improve the model performances. These findings demonstrated the suitability of the methods developed to discriminate geographical origin of lentils and confirmed the applicability of the infrared spectroscopy, in combination with chemometrics, to solve geographic origin issues of foodstuffs.

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.

The aim of this study was to analyse the transcriptional regulation of enniatins (ENs) production in Fusarium avenaceum. Methods and Results: We develop a new method to quantify ENs in FDM agar medium. We performed an LC/MS/MS analysis to evaluate enniatin A, A1, B, B1 and B4 production by seven F. avenaceum strains and, in a time-course experiment, by ITEM 3404 to analyse the transcriptional regulation of the esyn1 gene. The expression profile, achieved by Real time reverse transcriptase assay, showed an activation of gene transcription at the seventh day of incubation, corresponding to the higher increase of total ENs production. Enniatin B was the most abundant ENs analogues, representing the 90% of total ENs. The relative percentage of ENs remained unaltered during the experiment. Conclusions: We reported a transcriptional regulation of esyn1 responsible for the modulation of ENs biosynthesis. Significance and Impact of the Study: Enniatins are cyclic depsipeptides metabolites with a wide range of biological activities. They are also widespread contaminants in grains and cereals due to infection by enniatin-producing Fusarium species. This is the first article describing the transcriptional regulation of esyn1 gene that modulates ENs production in Fusarium avenaceum and provides new knowledge about the molecular mechanism underlying the biosynthesis of these important fungal metabolites in this toxigenic fungal species. © 2013 The Society for Applied Microbiology.

The importance and widespread incidence of Fusarium poae as a natural contaminant of wheat in differentclimatic areas warrants investigation into the genetic diversity and toxin profile of a northern Italy population.Eighty-one strains of F. poae isolated from durum wheat kernels, identified by species-specific polymerase chainreaction and translation elongation factor-1 gene sequence analysis, were genetically characterized by theamplified fragment length polymorphism (AFLP) technique and analysed by high-pressure liquid chromatographyfor their ability to produce the beauvericin (BEA) and trichothecene mycotoxins. A high level of variabilitywas observed by using AFLP analyses, with the lowest level of genetic similarity among the strains beingapproximately 61%. Most of the strains, 95%, produced BEA at52655 mg g1; 88% produced the trichothecenenivalenol at5865 mgg1 and 76% produced the trichothecene fusarenon-X at5167 mg g1. These data show thatF. poae can produce high amounts of BEA together with trichothecenes, and can represent a high potentialmycotoxin risk in Italy for wheat colonized by this species.

Highly efficient and eco-friendly antifungal fumigants are desirable in food and crop production. Although turmeric (Curcuma longa L.) essential oil is known to have a potent antifungal effect, the quality of whole essential oil can be unstable, leading to unreliable antifungal activities. The aim of the present study is to uncover the active individual compounds in turmeric essential oil that provide the antifungal properties using a convenient chemometric model. The Aspergillus flavus inhibition activities of essential oils derived from 24 batches of Curcuma longa L. were evaluated, with various fumigation concentrations from 50 ?L to 500 ?L essential oils per plate. Meanwhile, eighteen volatile compounds were identified by static headspace gas chromatography-mass spectrometry. To combine the antifungal activities and chemical profiles with the spectrum-effect relationship based on the partial least squares model, three volatile compounds (i.e., eucalyptol, beta-pinene and camphor) were identified and verified as the most potent antifungal compounds by their higher contribution factor to antifungal indexes. Thus, this research will provide a useful approach to screen bioactive compounds, and these three compounds are promising antifungal alternatives to conventional treatments for the control of A. flavus contamination.

Pleurotus eryngii (DC) Quél., comunemente noto con il nome di "fungo cardoncello", oltre ad essere un fungo edule molto apprezzato per le caratteristiche organolettiche e per il valore nutritivo, viene anche studiato per la capacità di produrre enzimi ligninolitici che hanno un elevato interesse biotecnologico. Il sistema enzimatico ligninolitico di P. eryngii, costituito dagli enzimi laccasi, lignino-perossidasi e Mn-perossidasi, ha un ruolo fondamentale nella demolizione della lignina in composti a basso peso molecolare. Poiché tali enzimi non hanno specificità di substrato, possono trovare impiego nella biodegradazione di molecole aromatiche complesse e di composti recalcitranti nell'ambito di applicazioni biotecnologiche come la decolorazione di coloranti sintetici e il biorisanamento ambientale da inquinanti chimici. Inoltre, alcuni recenti studi indicano che gli enzimi laccasi e Mn-perossidasi sono anche in grado di degradare l'aflatossina B1 (AfB1), una micotossina prodotta da funghi del genere Aspergillus nel corso di processi di ammuffimento di prodotti agricoli (specialmente mais) e la cui assunzione tramite alimenti contaminati può causare gravi patologie nell'uomo e in specie zootecniche. L'obiettivo di questo studio è stato quello di valutare la capacità degradativa dell'AfB1 da parte di diversi ceppi di P.eryngii. L'analisi cromatografica mediante Ultra Performance Liquid Chromatography (UPLC) dei filtrati colturali di 9 ceppi di P. eryngii coltivati in un substrato liquido a base di estratto di malto (MEB) con l'aggiunta di 500 ng/ml di AfB1 ha evidenziato la totale scomparsa dell'AfB1 dal brodo di coltura dopo 30 giorni. Gli stessi isolati, coltivati su substrato agarizzato a base di estratto di malto e contenente paglia di grano e farina di mais (MEASM), hanno mostrato una capacità detossificante compresa tra 65% e 84%, Un ceppo di P.eryngii, selezionato tra quelli più dotati di capacità degradativa, è stato infine coltivato su un substrato idoneo alla produzione dei carpofori e costituito da paglia di grano (50%), mais contaminato con Afb1 (128 ng/g, 25%), scarti della lavorazione della barbabietola da zucchero (12,5%) e favino (12,5%). Anche su questo substrato è stata osservata una detossificazione dell'86% dell'AfB1 dopo 28 giorni dall'inoculazione del fungo, in accordo con i risultati ottenuti in vitro. Nei corpi fruttiferi non è stata rilevata la presenza di AfB1, escludendo quindi un possibile trasporto della tossina nella parte edibile del fungo. L'efficienza biologica e la resa produttiva di carpofori non hanno mostrato differenze significative rispetto al controllo. I risultati di questo studio aprono possibilità di sviluppo di tecnologie per la conversione e la valorizzazione di prodotti agricoli contaminati con AfB1, che vengono attualmente distrutti e che potrebbero invece destinarsi alla produzione di mangimi ad uso zootecnico. Ulteriori studi sono in corso per identificare i prodotti di degradazione dell'AfB

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

The ability to produce fumonisin mycotoxins varies among members of the black aspergilli. Previously, analyses of selected genes in the fumonisin biosynthetic gene (fum) cluster in black aspergilli from California grapes indicated that fumonisin-nonproducing isolates of Aspergillus welwitschiae lack six fum genes, but nonproducing isolates of Aspergillus niger do not. In the current study, analyses of black aspergilli from grapes from the Mediterranean Basin indicate that the genomic context of the fum cluster is the same in isolates of A. niger and A. welwitschiae regardless of fumonisin-production ability and that full-length clusters occur in producing isolates of both species and nonproducing isolates of A. niger. In contrast, the cluster has undergone an eight-gene deletion in fumonisin-nonproducing isolates of A. welwitschiae. Phylogenetic analyses suggest each species consists of a mixed population of fumonisin-producing and nonproducing individuals, and that existence of both production phenotypes may provide a selective advantage to these species. Differences in gene content of fum cluster homologues and phylogenetic relationships of fum genes suggest that the mutation(s) responsible for the nonproduction phenotype differs, and therefore arose independently, in the two species. Partial fum cluster homologues were also identified in genome sequences of four other black Aspergillus species. Gene content of these partial clusters and phylogenetic relationships of fum sequences indicate that non-random partial deletion of the cluster has occurred multiple times among the species. This in turn suggests that an intact cluster and fumonisin production were once more widespread among black aspergilli.

Volatile metabolites from mold contamination have been proposed for the early identification of toxigenic fungi to prevent toxicological risks, but there are no such data available for Fusarium poae. F. poae is one of the species complexes involved in Fusarium head blight, a cereal disease that results in significant yield losses and quality reductions. The identification of volatile organic compounds associated with F. poae metabolism could provide good markers to indicate early fungal contamination. To this aim, we evaluated the volatile profile of healthy and F. poae-infected durum wheat kernels by SPME-GC/MS analysis. The production of volatile metabolites was monitored for seven days, and the time course analysis of key volatiles was determined. A total of 29 volatile markers were selected among the detected compounds, and multivariate analysis was applied to establish the relationship between potential volatile markers and fungal contamination. A range of volatile compounds, including alcohols, ketones, esters, furans and aromatics, were identified, both in contaminated and in healthy kernels. However, the overall volatile profile of infected samples and controls differed, indicating that the whole volatile profile, rather than individual volatile compounds, could be used to identify F. poae contamination of durum wheat grains.

Authentication of food is a burning topic and the development of rapid and reliable analytical strategies to authenticate the origin of food has become a priority at global level to combat food frauds. Food authentication is typically attained by applying the classical targeted approach where a certain analyte defined by characteristic parameters is further confirmed by reference standards. The non targeted approach offering the advantage of rapidity of the analysis without requiring any knowledge about the composition of the food sample to be analysed, has emerged in the last years gaining increasing attention and has been taken up by European research projects such as Food Integrity as objective of the WP18. Such approach allows to capture the highest number of information also referred to as features that are strictly correlated to the whole food matrix analysed. The comparison of food fingerprints to an authentic sample set will enable through the use of multivariate statistical models, detection of food sample adulteration or misdescription. However the bottleneck of such approach relies on the extensive data treatment required before submitting the pre-processed matrix to statistical analysis.In this work we present the optimization of a workflow based on the coupling between a Direct Analysis in Real Time (DART)ambient pressure source and a single cell Orbitrap(TM) based mass spectrometer applied to the non targeted analysis of foods to track the geographical origin and/or food integrity/authenticity.A typical workflow describing the main steps of a DART-HRMS analysis such as optimization of instrumental settings, sample preparation and post-acquisition data treatment to make the final data suitable for further statistical evaluation, will be presented along with the most critical steps. The pre-processing should include noise filtering, background subtraction, mass shift correction, mass alignment, spectra normalization, etc. Finally, an averaged spectrum representative of a certain food is typically generated from the analytical platform in use, that can be further converted into a format compatible for the subsequent statistical analysis. The duly optimization of all pre-processing steps on the acquired MS data is fundamental to produce reliable data prone to be submitted to multivariate statistic to generate trustful results. A case study reporting a typical DART-HRMS based workflow herein optimized will be shown applied to different food samples for food authenticity studies.

The invention relates to a method for table olive fermentation comprising the steps of: a. soaking the olives in brine;b. inoculating the product obtained in step a. with a yeast culture;c. incubating the product obtained in step b. in order to perform the alcoholic fermentation; d. inoculating the fermented product obtained in step c. using a Lactic Acid Bacterium (LAB) culture;e. incubating the product obtained in step d. to in order to perform the lactic fermentation.Procedures for the selection of starter cultures and their use in fermentation of two cultivars of table olives are described. Some claims are directed to specific starter cultures and the uses thereof for preparing fermented table olives.