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

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

Aspergillus section Nigri is an important group of species for food and medical mycology, and biotechnology. The Aspergillus niger 'aggregate' represents its most complicated taxonomic subgroup containing eight morphologically indistinguishable taxa: A. niger, Aspergillus tubingensis, Aspergillus acidus, Aspergillus brasiliensis, Aspergillus costaricaensis, Aspergillus lacticoffeatus, Aspergillus piperis, and Aspergillus vadensis. Aspergillus awamori, first described by Nakazawa, has been compared taxonomically with other black aspergilli and recently it has been treated as a synonym of A. niger. Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins ?-tubulin (benA), calmodulin (CaM), and the translation elongation factor-1 alpha (TEF-1?) of a population of A. niger strains isolated from grapes in Europe revealed the presence of a cryptic phylogenetic species within this population, A. awamori. Morphological, physiological, ecological and chemical data overlap occurred between A. niger and the cryptic A. awamori, however the splitting of these two species was also supported by AFLP analysis of the full genome. Isolates in both phylospecies can produce the mycotoxins ochratoxin A and fumonisin B2, and they also share the production of pyranonigrin A, tensidol B, funalenone, malformins, and naphtho-?-pyrones. In addition, sequence analysis of four putative A. awamori strains from Japan, used in the koji industrial fermentation, revealed that none of these strains belong to the A. awamori phylospecies.

Branch cankers and stem-end rot are two of the most important threats to avocado production. During the autumn of 2013, sampling was conducted in the main avocado growing area in eastern Sicily to study the occurrence and establish the causal agents of branch canker and stem-end rot. A total of 94 fungal isolates, recovered from four avocado orchards, were identified by morphological characterisation, DNA sequencing and phylogenetic analyses as belonging to the genera Colletotrichum, Neofusicoccum or Diaporthe. The majority of the isolates were identified as Neofusicoccum parvum (70.2 %), with the remaining isolates being Colletotrichum gloeosporioides or C. fructicola (16 %), and Diaporthe foeniculacea or D. sterilis (13.8 %), respectively. Pathogenicity tests showed N. parvum was the most virulent species (P = 0.05), whereas Diaporthe isolates were the least so. An intermediate virulence was observed for C. gloeosporioides and C. fructicola, which were associated only with stem-end rot of fruit. Regarding cultivar susceptibility of fruit to these pathogens, 'Hass' was more susceptible to infection by C. fructicola and D. foeniculacea compared with 'Bacon' whereas no significant differences were detected for the remaining pathogens. To our knowledge, this is the first account of the pathogens causing branch canker and stem-end rot of avocado in Italy, and the first studies comparing the relative virulence of each species involved

Anthracnose symptoms consisting of necrotic spots on the leaves, twigs and branches were observed on mango trees of cv. Kensington Pride in orchards located in the countryside of Palermo and Milazzo (southern Italy). Based on morphological observations and phylogenetic analysis of the ?-tubulin (benA) and histone H3 (HIS3) genes, three Colletotrichum species were identified and recovered from diseased plants, i.e. C. karstii (nine isolates), C. kahawae subsp. ciggaro (six isolates) and C. gloeosporioides (six isolates). Following artificial inoculation, all species induced symptoms on the leaves and fruits of cv. Kensington Pride. To our knowledge, this is the first report of mango anthracnose caused by C. karstii, C. kahawae subsp. ciggaro and C. gloeosporioides in Italy.

The Sicilian coasts provide suitable environmental conditions for production of high-quality tropical and subtropical fruits. In particular, avocado (Persea americana) and mango (Mangifera indica) orchards increased in last years on this area. Postharvest infections of tropical and subtropical fruits commonly occurs wherever the crops are cultivated. Several fungal species are reported as causal agents of anthracnose and stem-end rot. Among these, Botryosphaeria spp. and Colletotrichum spp. are the mostly spread worldwide. In Mediterranean environment, decays caused by several fungal pathogens are reported on plants and fruits of mango, but extensive surveys on avocado orchards were never done. The aims of this study were to determine the occurrence of stem-end rot disease in one of the major avocado growing areas in southern Italy and to identify the fungal species associated with fruits symptoms basing on morphological and molecular analysis. Approximately 100 avocado fruits cv. "Hass" were collected in four orchards in Catania province and incubated in laboratory. Stem-end rot developed from 5 to 10 days. Small pieces of symptomatic flesh from the margin of infected area were placed onto potato dextrose agar. A total of 47 isolates were recovered. Conidia characteristics and colony morphology were determined. Multilocus sequences analysis was performed using beta-tubulin gene, internal transcribed spacers of the ribosomal DNA and translation elongation factor gene (for Botryosphaeria spp.) or histone 3 gene (for Colletotrichum spp.). The molecular analysis allowed the identification of 68% of isolates as Neofusicoccum parvum, 17% as Colletotrichum gloeosporioides and 15% as C. fructicola. To our knowledge, these are the first data on occurrence of N. parvum, C. gloeosporioides and C. fructicola associated with stem-end rot of avocado in Europe. Further studies on pathogenicity ability of these species, in pre and postharvest conditions, should be carried out.

During the 2012 and 2013 growing seasons, a disease was detected on potted laurustinus (Viburnum tinus) plants in two nurseries located in the Catania province (eastern Sicily, Italy). 'Cylindrocarpon'-like species were consistently recovered from crown rot and stem rot tissues. Based on morphological characteristics, DNA sequencing and phylogenetic analysis of ?-tubulin (TUB), histone H3 (HIS3) and translation elongation factor-1? (TEF-1 ?) gene sequences, the fungi associated with symptomatic tissues were identified as 'Cylindrocarpon' pauciseptatum, Ilyonectria novozelandica and I. torresensis. Koch's postulates were fulfilled by pathogenicity tests carried out on potted V. tinus cuttings. To our knowledge, this is the first report worldwide of 'C.' pauciseptatum, I. novozelandica and I. torresensis causing disease on V. tinus. © 2014 Blackwell Verlag GmbH.

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

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

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

L'interesse per l'ozono quale agente sanitizzante nell'industria alimentare è aumentato negli ultimi anni in risposta ad una sempre crescente richiesta di una 'chimica verde'. L'ozono è infatti un composto rispettoso dell'ambiente in quanto si decompone rapidamente in ossigeno e non lascia residui negli alimenti. Tale gas è considerato un additivo alimentare GRAS (Generally Recognised As Safe) ed il suo utilizzo come additivo antimicrobico per il contatto diretto con gli alimenti è stato recentemente approvato dalla Food and Drug Administration (FDA). Recenti studi hanno mostrato come l'ozono sia efficace nel controllo di insetti, batteri e funghi e nel degradare pesticidi e micotossine che possono contaminare i cereali. Scopo del presente studio è stato quello di valutare l'effetto dei trattamenti con ozono gassoso a diverse concentrazioni (9,0-15,4-26,1 g/m3) e tempi di contatto (2-8-12-24 ore) sulla contaminazione da funghi filamentosi, lieviti e micotossine (in particolare deossinivalenolo e tossine T-2 e HT-2) in campioni di frumento duro utilizzando un prototipo di generatore di ozono progettato ad hoc per il trattamento delle cariossidi. E' stato inoltre valutato l'effetto dei trattamenti su alcuni parametri di qualità del frumento, in particolare sul contenuto in ceneri, proteine, amido, fibra, glutine e indice di giallo. I trattamenti con ozono alle concentrazioni di 9,0 e 15,4 g/m3 non hanno evidenziato effetti significativi sulla contaminazione da funghi filamentosi e lieviti per tutti i tempi di contatto, rispetto al controllo non trattato. In tali condizioni operative è stata osservata una riduzione del contenuto di DON (fino al 19%), rispetto al controllo non trattato, già a partire dalle 8 ore di contatto. I trattamenti con ozono a concentrazioni maggiori (26,1 g/m3) hanno determinato sia una riduzione significativa della carica microbica già a partire dalle 2 ore, sia una maggiore riduzione del contenuto di DON, fino al 32%, a partire dalle 12 ore di trattamento. Non è stata invece osservata alcuna variazione significativa del contenuto di tossine T-2 e HT-2 per tutti i trattamenti. Nelle diverse condizioni sperimentali non sono state osservate variazioni significative dei parametri qualitativi del frumento.

Il deossinivalenolo (DON), prodotto da diverse specie di Fusarium, si ritrova frequentemente come contaminante naturale nel frumento e in altri cereali. Spesso lo si ritrova associato a livelli significativi della sua forma modificata DON-3-glucoside (DON-3-Glc). L'ozono (O3) è un potente disinfettante e ossidante classificato come GRAS (Generalmente riconosciuto come sicuro). Esso reagisce facilmente con molti composti specifici compreso le micotossine, degradandole in soluzione acquosa, per cui ha il potenziale per essere efficace anche per la decontaminazione dei grani. In questo studio sono riportati i risultati sull'efficacia di trattamenti con ozono gassoso per la riduzione di DON, DON-3-Glc, batteri, funghi e lieviti in frumento duro contaminato naturalmente. Per le prove di ozonazione è stato usato un prototipo costituito da un cilindro rotante, contente il campione di cariossidi da trattare, in cui veniva insufflato ozono gassoso a diverse concentrazioni e tempi di esposizione. Sono state identificate le condizioni ottimali (55 gO3 h-1 per 6 h) che erano efficaci nel diminuire i livelli di contaminazione del frumento duro senza alterare i parametri chimici e reologici del frumento trattato, della semola e pasta da esso ottenuti. Le riduzioni medie di DON e DON-3-Glc nel grano ozonato erano rispettivamente del 29 e del 44%. L'ozonazione ha inoltre prodotto una riduzione significativa (p <0,05) del conteggio totale (CFU/g) di batteri, funghi e lieviti.

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

During surveys conducted in 2010-2013, a complete breakage or bending of the trunk and a dry basal stem rot were observed on containerised Brahea armata, B. edulis, Howea forsteriana and Trachycarpus princeps plants in different nurseries located in eastern Sicily (southern Italy). A cylindrocarpon-like species was consistently obtained from diseased palm tissues, while known pathogens of these hosts such as Ganoderma, Phytophthora and Thielaviopsis were not found associated with symptomatic tissues or isolated on standard or selective media. A total of 40 cylindrocarpon-like isolates were collected and characterised based on morphology and DNA phylogeny. Multigene analyses based on the beta-tubulin, histone H3, translation elongation factor 1-alpha, and the internal transcribed spacers (ITS1, 5.8S, ITS2) genes facilitated the identification of a new species, described here as Ilyonectria palmarum. The pathogenicity of one representative isolate collected from each palm species was tested on plants cultivated under nursery conditions and in a growth chamber. All isolates were pathogenic to B. armata, B. edulis, H. forsteriana, and T. princeps and symptoms identical to that observed in nurseries were reproduced. Dry basal stem rot and stem bending caused by Ilyonectria palmarum represents a potentially serious problem for nurseries cultivating containerised palms.

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/).

hirty-two isolates belonging to black aspergilli (Aspergillus section Nigri) associated to vine canker disease of grapevine were collected in seven vineyards located in southeastern Sicily (Italy). Molecular analysis was performed to identify the isolates by multilocus sequence analysis. Amplification of part of the ?-tubulin gene (benA) and partial calmodulin (CaM) gene were performed using the Bt2a, Bt2b and CL1, CL2A primers, respectively. Molecular characterisation showed a high distribution of Aspergillus niger "aggregate" species on grapes in Sicily and in particular of A. niger (21 isolates), A. tubingensis (9 isolates), and A. carbonarius (2 isolates). The 21 isolates of A. niger found to belong within the newly described cryptic species A. awamori. Six isolates (3 of A. tubingensis, 2 of A. carbonarius, and 1 of A. niger) were used in pathogenicity studies on mature canes of cv. Italia grape. All species caused Aspergillus vine canker equally well, with no differences in virulence.

Maximum permitted levels for aflatoxins in dried fruits such as almonds and apricot kernels are higherin kernels intended for further processing as compared to ready-to-eat derived products. The effect ofelectronic sorting, peeling and manual colour sorting on apricot kernels was tested to check levels anddistribution of aflatoxins. The fate of aflatoxins during transformation processes of almonds into nougat,pastries and almond syrup (blanching, roasting, water infusion and cooking) was also evaluated. Themass balance approach was used to determine levels and distribution of aflatoxins in each fractioncollected during processing steps. Experiments were conducted on naturally contaminated apricotkernels and almonds inoculated with a toxigenic strain of Aspergillus flavus. An improved HPLC-FLDmethod was used for aflatoxin determination in all the matrices considered in this study. Highly variableresults were obtained with electronic sorting experiments because rejected fractions contained 13-59%of total aflatoxins. The manual colour sorting of peeled apricot kernels gave excellent results because theremoval of discoloured kernels removed 97.3-99.5% of total aflatoxins. Blanching processes (bysteaming or boiling in water) did not reduce aflatoxin levels in blanched almonds and apricot kernelswhereas the removal of skins removed only 7-10% of total aflatoxins. Negligible amounts of aflatoxins(<1%) were found in boiling water analysed after blanching contaminated almonds or apricot kernels.Almond pastries were prepared by mixing almond paste, eggs and sugar that were cooked at 140°C for30 min, 160°C for 20 min or 180°C for 15 min. Aflatoxins were substantially stable during preparationand cooking of pastries and a slight reduction of aflatoxins (10%) was observed only at 180°C. For thepreparation of nougat the peeled almonds were roasted at 150°C for 30 min then mixed with caramel orcaramel+honey at 105°C until cooked, then shaped into small heaps. Roasting produced about 50%reduction of aflatoxins. Higher aflatoxin reduction (82%) was observed by roasting at 150°C for 120min, but almonds lost their organoleptic characteristics. The preparation and cooking of nougat produceda further consistent reduction of 54-70% of aflatoxins. Almond syrup was prepared from peeled almondpaste that was infused in water for 5 hours. After infusion the exhausted almonds were discarded and theinfuse was sugared and boiled until reaching the consistency of syrup. The whole process of almondsyrup preparation produced a marked increase of total aflatoxins probably due to the involvement ofenzymes during the infusion step that released free aflatoxins from masked aflatoxins. About 15-22% oftotal aflatoxins passed in the final syrup during the whole process of almond syrup preparation. Thesenew information could be useful for food producers to keep aflatoxin contamination under control andimprove the safety of almond

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

Deoxynivalenol (DON) occurs frequently in wheat grains and it is often associated with significant levels of its modified form DON-3-glucoside (DON-3G). Several approaches were tested in the past to prevent the formation of DON in the field or reduce its level in the grains. Ozone (O3) is a powerful disinfectant and oxidant that reacts quite easily with specific compounds including mycotoxins and pesticides. It was demonstrated to degrade these contaminants in aqueous solution and it has the potential to be also effective for decontamination of grains. Ozone in gaseous form as a fumigant gave also promising results against insects and a wide range of microorganisms including bacteria, fungi, viruses, protozoa and fungi spores that may grow and survive on wheat during storage. Moreover, it is classified as GRAS (Generally Recognised As Safe) which makes very attractive its use in food industry. However, ozone is unstable and decays naturally into inactive diatomic oxygen (O2) and it must be continuously added in the mass of the grain to maintain the active concentrations. We have tested the efficacy of gaseous ozone treatments for reduction of mycotoxins, pesticides, heavy metals, bacteria, fungi and yeasts in durum wheat grain naturally contaminated with low levels of these contaminants. A prototype was tested to continuously and homogeneously dispense ozone in 2 kg aliquots of durum wheat. Levels and distribution of contaminants were measured before and after ozone treatments (different concentrations and time of exposure), as well as in all fractions obtained during wheat processing. The optimal conditions of ozone treatments were identified in order to maximize the reduction of contaminants and maintain unaffected chemical and rheological parameters of durum wheat. Therefore, optimal ozonation conditions were applied to 20 kg of durum wheat that were successively processed into semolina and pasta. A significant reduction of the levels of some contaminants was obtained, with minor influence on qualitative parameters of durum wheat. The scale up at industrial level for ozone treatment of wheat grains will be discussed.

Fungi have an important role in the production of dry-cured meat products, especially during the seasoning period. In general, both industrially and handmade salami are quickly colonized by a composite mycobiota during seasoning, often with a strong predominance of Penicillium species. These species are involved in the improvement of the characteristics and taste, and in the prevention of the growth of pathogenic, toxigenic or spoilage fungi. During the survey of fungal species occurring on the salami surface and in the air of the seasoning and storage areas of a salami plant (Calabria, Italy), two Penicillium species were predominantly present. One species was identified as Penicillium nalgiovense, and the other was related to, but distinct from, Penicillium olsonii. Further molecular and biochemical analyses showed that this strain has high homology with the not yet described species named ". Penicillium milanense" isolated in Denmark and Slovenia on cured meats. The taxonomic position of these strains in Penicillium was investigated using calmodulin, ? tubulin and ITS sequences, phenotypic characters and extrolite patterns, and resulted in the discovery of a new Penicillium species, described here as P. salamii. A literature search showed that this species occurs on (cured) meat products worldwide. In our study, P. salamii predominated the salami and capocollo surface in levels similar to the commonly known starter culture P. nalgiovense, irrespective of the room or age of seasoning. Preliminary inoculation trials with P. salamii showed that it was able to colonize salami during seasoning, indicating that this species could be used as a fungal starter for dry-cured meat.

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.

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

Calonectria species have been reported as devastating pathogens mostly on horticultural and forest crops worldwide. Since these pathogens represent a serious threat for the nursery production, the aim of this study was to investigate on the short-term potential of soil solarization for eradicating Calonectria microsclerotia.MethodsTwenty Calonectria isolates collected in Italy from different hosts and locations were identified by using DNA sequencing of ?-tubulin. The effect of thermal regimes and innovative solarizing films on the soil survival of Calonectria microsclerotia was evaluated through time at different sampling periods in growth chamber and greenhouse experiments.ResultsEleven and nine isolates were identified as Calonectria pauciramosa and Calonectria polizzii, respectively. No viable Calonectria inoculum was recovered after 12 days from all solarized plots inside ethylene-tetrafluoroethylene (ETFE) greenhouse and at 15-cm depth from solarized plots inside ethylene-vinyl-acetate (EVA) greenhouse. Under EVA cover, solarization killed C. pauciramosa microsclerotia within 9 and 17 d at 15- and 30-cm depths in soil, respectively, whereas no viable inoculum was retrieved within 6 and 12 days from solarized plots inside ETFE greenhouse.ConclusionsThis paper demonstrates that short-term soil solarization is effective for Calonectria microsclerotia suppression in nurseries, and shows that ETFE film as well as other innovative materials could improve this technique.

Penicillium nordicum, an important and consistent producer of ochratoxin A (OTA), is a widely distributed contaminant of NaCl and protein rich food. It is usually found on dry-cured meat products and is considered the main responsible of their contamination by OTA. The aim of this work was to study the gene expression of a polyketide synthase (otapksPN), involved in P. nordicum OTA biosynthesis, and OTA production during a small-scale seasoning process. Fresh pork sausages were surface inoculated with P. nordicum and seasoned for 30 days. Gene expression and OTA production were monitored throughout the seasoning process after 4, 5, 6, 7, 10, 14, and 30 days. The expression of otapksPN gene was already detected after 4 days and increased significantly after 7 days of seasoning, reaching the maximum expression level after 10 days (1.69·104 copies/100 mg). Consistently with gene expression monitoring, OTA was detected since the 4th dayand its content increased significantly from the 7th day, reaching the maximum level after 10 days. In the late stages of seasoning process, OTA did not increase further and the number of gene copies was progressively reduced after 14 and 30 days.

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.