Introduction. Fungi (yeasts and moulds) are recognized as one of the main contaminants of dairy products including yogurt and sour milk. These microorganisms can also cause spoilage in a wide range of processed, preserved and refrigerated food products. During the past years, several molecular methods based on immunological and genotypic techniques have been developed for revealing the presence of undesirable microorganisms, including fungi, in different food matrices. However, no commercial kit are already available to detect viable yeasts and moulds in dairy products. Materials and methods. Five antibodies against yeasts and molds were selected from commercially available antibodies and used to produce functionalized magnetic beads to be used to capture and separate microrganisms associated to dairy products. Four yeast type species (Debaryomyces hansenii, Kluyveromyces marxianus, Geotrichum candidum and Pichia anomala) and four mold species (Alternaria alternata, Aspergillus niger, Penicillium italicum and Rhizopus stolonifer) were used. Milk, yogurt and soft cheese were tested as matrices. A RT-PCR protocol was developed for detection of yeast and molds mRNA extracted from contaminated foods. Results. A new method for yeast and molds enrichment from different food dairy products (milk, yogurt and soft cheese) based on the use of antibody coated magnetic beads was developed. A new RT-PCR assay based on a nested amplification was optimized for the detection of yeast and molds in artificially contaminated dairy products.Discussion. The correlation between the amplification signal and the microbial count will allow to use this method for viable contaminants quantification in dairy products. This method can avoid the labor expensive food matrices treatments, often cause of loss of sensitivity. This approach will be transferred also in other food matrices. This procedure can be implemented by the use of automated enrichment systems already available for pathogen microorganisms.

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 is one of the economically most important fungal genera. Recently, the ICN adopted the single name nomenclature which has forced mycologists to choose one name for fungi (e.g. Aspergillus, Fusarium, Penicillium, etc.). Previously two proposals for the single name nomenclature in Aspergillus are presented: one attributes the name "Aspergillus" to clades comprising seven different teleomorphic names, by supporting the monophyly of this genus; the other proposes that Aspergillus is a non-monophyletic genus, by preserving the Aspergillus name only to species belonging to subgenus Circumdati and maintaining the sexual names in the other clades. The aim of our study was to test the monophyly of Aspergilli by two independent phylogenetic analyses using a multilocus phylogenetic approach. One test was run on the publicly available coding regions of six genes (RPB1, RPB2, Tsr1, Cct8, BenA, CaM), using 96 species of Penicillium, Aspergillus and related taxa. Bayesian (MrBayes) and Ultrafast Maximum Likelihood (IQ-Tree) and Rapid Maximum Likelihood (RaxML) analyses gave the same conclusion highly supporting the monophyly of Aspergillus. The other analyses were also performed by using publicly available data by using the coding sequences of nine loci (18S rRNA, 5,8S rRNA, 28S rRNA (D1-D2), RPB1, RPB2, CaM, BenA, Tsr1, Cct8) of 204 different species. Both Bayesian (MrBayes) and Maximum Likelihood (RAxML) trees obtained by this second round of independent analyses strongly supported the monophyly of the genus Aspergillus. The stability test also confirmed the robustness of the results obtained. In conclusion, statistical analyses have rejected the hypothesis that the Aspergilli are non-monophyletic, and provided robust arguments that the genus is monophyletic and clearly separated from the monophyletic genus Penicillium. There is no phylogenetic evidence to split Aspergillus into several genera and the name Aspergillus can be used for all the species belonging to Aspergillus i.e. the clade comprising the subgenera Aspergillus, Circumdati, Fumigati, Nidulantes, section Cremei and certain species which were formerly part of the genera Phialosimplex and Polypaecilum. Section Cremei and the clade containing Polypaecilum and Phialosimplex are proposed as new subgenera of Aspergillus.The phylogenetic analysis also clearly shows that Aspergillus clavatoflavus and A.zonatus do not belong to the genus Aspergillus. Aspergillus clavatoflavus is therefore transferred to a new genus Aspergillago as Aspergillago clavatoflava and A. zonatus was transferred to Penicilliopsis as P. zonata. The subgenera of Aspergillus share similar extrolite profiles indicating that the genus is one large genus from a chemotaxonomical point of view. Morphological and ecophysiological characteristics of the species also strongly indicate that Aspergillus is a polythetic class in phenotypic characters.

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.

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.

Microorganisms are essential components of biological diversity, fundamental elements which guarantee the existence of sustainable ecosystems. About 50% of the living biomass on the planet is microbial and microorganisms provide an important source of genetic information for molecular biology and biotechnology which is being used in several business applications. Therefore it is of key economic importance to collect, store and characterise these microorganisms that can be used for several purposes. The role of the first microbial culture collection (CBS in the NL), apart from research purposes, was related to agriculture, brewing and medicine. In 1930, with the discovery of penicillin, the importance of these collections strongly raised together with the awareness that fungi are great sources of biological activities. The industrial and biotechnological applications of fungi include brewing and wine making, baking food processing, enzyme productions, antibiotics, organic acid and vitamin production, genetic engineering, pesticides and insecticides development. The increasing number of culture collections worldwide, private or institutional, mirrors the need to preserve this wealth protecting the microbial gene pool for biological researches, industrial applications and biodiversity preservation.Microbials and microorganisms group several types of organisms of which the fungi. Fungi that are moulding agents of agri-food products include species from the genera Aspergillus, Penicillium, Fusarium, Alternaria and Claviceps whose ability to form mycotoxins - substances responsible for repeated episodes of food poisoning both in livestock and in humans - is the cause of justified apprehension for food safety. This toxigenicity is unevenly distributed among species within a given fungal genus, and within the same species only some strains will have it, each usually having its own toxigenic profile. The need to assess this biodiversity, which is a requirement for the safety and protection of foodstuffs, led to the foundation of specific collections (or fungal reference material) and to research into new molecular diagnostic methods. These are the main objectives of the ITEM collection in Bari.In that context, the objective of this case study is to describe and identify added values and bottlenecks being faced by the ITEM Microbial Culture Collection in Bari, Italy which is recognised as a EU key factor in the conservation and sustainable use of agro-food toxigenic fungi. Additionally, the study analyses how this large collection has been used to develop new products and supported the development of business applications in the food sector and more particularly in the seasoning of salami in Italy, as well as in yeast and bacteria of winery fermentation of Apulia Region.

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.

Competitive exclusion by atoxigenic strains of Aspergillus flavus is worldwide accepted as the only preventive action, effective to minimize aflatoxins contamination in several crops, maize included. Maize lots, uncompliant with the European legislation, were signaled almost all years. After the first outbreak of aflatoxin in maize in 2003 in Italy, followed by severe contamination in 2012 and 2015, but lot uncompliant with the European legislation signaled almost all years, the selection of candidate biocontrol agents among native strains started. USA, Africa and Italy have now commercial products available, but Eastern Europe need to be considered too.The aim of this study was select A. flavus candidate biocontrol agents in Romania. For this purpose, 139 maize flour samples, representative of the whole Romanian maize growing areas, were used for the isolation of Aspergillus section Flavi strains. Among them, 188 representative strains were chosen, identified (sequencing the beta-tubulin gene region), processed and characterized. Deletions of the aflatoxin gene cluster region were investigated by multiplex PCR analyses (using a set of 16 primer pairs) to select potential biocontrol agents to be included in competition tests. The size of microsatellite alleles retrieved from 18 markers, out of the 24 tested, were bioinformatically analysed to uncover the population structure. 169 strains were confirmed as A. flavus; 71 strains seems to lack at least one gene of the aflatoxin cluster and, among these, 7 strains seems to lack all the genes and were chosen for further investigations. Microsatellites analysis allowed us to identify two main groups by bayesian cluster analysis, genetic distance based analyses and population assignment. The 7 atoxigenic strains demonstrated to be very effective in reducing aflatoxins (ranged between 80 and 96%) when co-inoculated with toxigenic strains. A potential candidate to be used as biocontrol agent was finally selected on the basis of competition tests and microsatellites results and, its efficacy, will be hopefully confirmed during in field trials.

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.

Ochratoxin A (OTA) is a potent pentaketide nephrotoxin diffusely distributed in food and feed products (grains, legumes, coffee, dried fruits, meat derived products, beer and wine); it is also carcinogenic, neurotoxic, teratogenic and immunotoxic. This mycotoxin is produced by species belonging to the genus Aspergillus and Penicillium. OTA is the primary mycotoxin risk in wine and dried vine fruits, its maximum level is regulated by law. Several studies focused on Aspergillus section Nigri, due to their role as causative agents of black rot of grapes, and subsequently as cause of ochratoxin A contamination. In particular, Aspergillus carbonarius has been identified as the major cause of contamination in grape berries. This contamination is strongly related to climatic conditions, geographical regions, grape varieties, damage by insects, growing season; in particular great variations may occur from one year to another. So, climate represents an important key-factor in the agro-ecosystem, influencing fungal colonization and ochratoxin A production in grapes. Climate change is expected to have a profound effect on our landscape worldwide, and also to have an important impact on sustainable food production system. Recent studies have reported how the climate change may affect mycotoxins production in the fields and the relevant risk on economically important crops. In this regard, the interacting effect of water stress (aw 0.99-0.93) and different day/night climate conditions simulating nowadays (18-31 and 15-28°C) and climate change scenarios (18-34 and 20-37 °C) in high OTA risk area of Southern Italy during the ripening season, were studied. Mycelial growth rate, OTA production and molecular expression of key genes (PKS, NRPS, Hal, p450, bZIP) of OTA biosynthetic cluster by A. carbonarius ITEM 5010 were measured. Our results showed that, in water stress conditions (0.93 aw), no OTA production was observed and, except at 20-37°C, the growth rate was slower compared to 0.99 aw. A significantly higher amount of OTA was observed at 0.99 aw and 18-34°C climate change scenario. Gene expression, monitored by quantitative real time RT-PCR, gave evidence of the high expression levels of OTA biosynthetic genes in this condition, in particular NRPS and Hal genes were strongly expressed. These preliminary and new results on A. carbonarius in a climate change scenario suggest that a possible slight increase of temperature may lead to higher OTA contamination and to a possible expansion of the risk area in the Mediterranean basin.

Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.ResultsHere we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.ConclusionsThe data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

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.

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.

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.

Aflatoxins can be found as contaminants in a wide range of foods, such as nuts, cereals, dried fruits and milk. Due to their hepatotoxic and carcinogenic effects, the maximum allowed concentration ofaflatoxins is nowadays regulated in many countries, with levels up to 50 ?g/kg. In routine analysis, the main methods used to determine aflatoxins are based on high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) [1]. Despite the very high sensitivity of these methods, they are destructive, expensive, time consuming and not appropriate for real time control, e.g., online. Consequently, the development of fast, non destructive and economic methods for aflatoxins detection and monitoring in food industry is becoming more and more important.Our studies showed that manual sorting of dark or spotted apricot kernels removed 97.3-99.5% of total aflatoxins [2]. However, discolored seeds could be visually identified only after removing theskins from each seed by means of a time-consuming operation. For these reasons, in this work we investigated the possibility to use NIR-HSI for the fast and nondestructive automated identification of aflatoxin contaminated unpeeled apricot kernels.On the whole 9 hyperspectral images, each one containing 48 kernels, were acquired in the 900-1700 nm range. After image acquisition, the kernels were peeled to identify the dark or spotted kernels and subjected to HPLC analysis for AFB1 quantification. Classification models were then calculated on a training set of NIR spectra extracted from a representative number of non-contaminated and dark seeds, selected on 5 images on the basis of HPLC analysis results as well as of the visual evaluation of the peeled kernels. The remaining 4 images were instead used as independent test set for model validation. Since dark seeds were found to have a higher concentration of AFB1 than spotted seeds, the latter ones were not included in the training set.Different iPLS-DA classification models, built using different signal preprocessing methods and different interval size values, were then evaluated in terms of classification efficiency in cross validation of the training set pixels, in order to select the optimal conditions. The results were reported under the form of predicted probability maps, and for each single kernel the contamination was estimated as the percentage of pixels assigned to the "contaminated" class by the iPLS-DA model.

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.

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.

The fate of aflatoxins during processing of contaminated almonds into nougat, pastries, and almond syrup was evaluated by testing the effect of each processing step (blanching, peeling, roasting, caramelization, cooking, and water infusion) on the distribution and levels of aflatoxins. Blanching and peeling did not reduce total aflatoxins that were distributed between peeled almonds (90-93%) and skins (7-10%). Roasting of peeled almonds reduced up to 50% of aflatoxins. Up to 70% reduction of aflatoxins was observed during preparation and cooking of almond nougat in caramelized sugar. Aflatoxins were substantially stable during preparation and cooking of almond pastries. The whole process of almond syrup preparation produced a marked increase of total aflatoxins (up to 270%) that were distributed between syrup (18-25%) and spent almonds (75-82%). The increase of total aflatoxins was probably due to the activation of almond enzymes during the infusion step that released free aflatoxins from masked aflatoxins.

Deoxynivalenol (DON) is an important mycotoxin produced by several species of Fusarium. It occurs often in wheat grain and is frequently associated with significant levels of its modified form DON-3-glucoside (DON-3-Glc). Ozone (O3) is a powerful disinfectant and oxidant, classified as GRAS (Generally Recognised As Safe), that reacts easily with specific compounds including the mycotoxins aflatoxins, ochratoxin A, trichothecenes and zearalenone. It degrades DON in aqueous solution and can be effective for decontamination of grain. This study reports the efficacy of gaseous ozone treatments in reducing DON, DON-3-Glc, bacteria, fungi and yeasts in naturally contaminated durum wheat. A prototype was used to dispense ozone continuously and homogeneously at different concentrations and exposure time, in 2kg aliquots of durum wheat. The optimal conditions, which do not affect chemical and rheological parameters of durum wheat, semolina and pasta, were identified (55g O3h-1 for 6h). The measured mean reductions of DON and DON-3-Glc in ozonated wheat were 29% and 44%, respectively. Ozonation also produced a significant (p<0.05) reduction of total count (CFU/g) of bacteria, fungi and yeasts in wheat grains.

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

The effect of triazole-based treatments on Fusarium head blight (FHB), grain yields and the accumulation of deoxynivalenol (DON) in harvested wheat kernels was evaluated by means of twenty multi-site field experiments performed during five consecutive growing seasons (from 2004-2005 to 2008-2009) in Italy. Fungicide treatments were carried out on different cultivars of common wheat (cv. Serio, Blasco, Genio and Savio) and durum wheat (cv. Orobel, Saragolla, San Carlo, Levante, Duilio, Karur and Derrik) after artificial inoculation with a mixture of toxigenic Fusarium graminearum and F. culmorum strains. The application of fungicides containing prothioconazole (Proline® or Prosaro®) at the beginning of anthesis (BBCH 61) resulted in a consistent reduction of FHB disease severity (by between 39 and 93%) and DON levels in wheat kernels (by between 40 and 91%) and increased wheat yields (from 0.4 to 5.6 t ha-1, average 2.2 t ha-1), as compared to the untreated/inoculated control. Fungicides containing tebuconazole (Folicur® SE) and cyproconazole plus prochloraz (Tiptor® Xcell) showed a reduced effectiveness compared with prothioconazole-based treatments. All fungicide treatments were more effective in reducing DON and increasing grain yields of common wheat than durum wheat. Results showed that the application of fungicides containing prothioconazole at the beginning of anthesis provided a strong reduction of FHB disease, allowing both an increase in grain yields and a considerable reduction of DON content in wheat kernels.

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

Il deossinivalenolo (DON) è una micotossina importante che si ritrova spesso nelle granaglie ed è frequentemente associato alla sua forma modificata DON-3-glucoside (DON-3-Glc). L'ozono (O3) è un potente disinfettante e ossidante che degrada il DON in soluzione acquosa e potrebbe essere efficace se usato direttamente sulle cariossidi. Questo studio riporta l'efficacia dei trattamenti con ozono gassoso nel ridurre DON, DON-3-Glc, batteri, funghi e lieviti in grano duro naturalmente contaminato. Un prototipo è stato utilizzato per erogare l'ozono in modo continuo e omogeneo a diverse concentrazioni e tempi di esposizione. Sono state identificate le condizioni ottimali che non influiscono sui parametri chimici e reologici del grano duro, della semola e della pasta (55 g di O3 h-1 per 6 ore). L'ozonizzazione ha prodotto una riduzione significativa (p<0,05) del conteggio totale (CFU/g) di batteri, funghi e lieviti, di DON (29%) e DON-3-Glc (44%).

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.

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.

Management of Calonectria spp. infections in nurseries requires scheduled fungicide applications, particularly with methyl benzimidazole carbamates (MBCs) and sterol demethylation inhibitors (DMIs). Due to rising concerns about the occurrence of MBC resistance in different Calonectria populations and variability in prochloraz efficacy in controlling these pathogens, a detailed study on prochloraz sensitivity distributions of Calonectria isolates belonging to the Calonectria scoparia complex was carried out. In total, 105 isolates collected in two distinct periods (1993 to 1996 and 2005 to 2009) were analyzed for prochloraz sensitivity. Based on DNA sequencing and phylogenetic analyses of ?-tubulin, histone H3, and translation elongation factor-1? gene sequences, 69 and 36 isolates were identified as C. pauciramosa and C. polizzii, respectively. The isolates collected more recently (group B) had a reduced prochloraz sensitivity, as indicated by greater values for the effective dose to reduce growth by 50% than those collected earlier (group A). The reduced sensitivity detected in vitro corresponded to partial loss of fungicide efficacy in controlling infections in red clover and feijoa under controlled and semi-field conditions, respectively. Frequent prochloraz application in nurseries for controlling Calonectria spp. infections is discouraged.

In May 2015, symptoms of light brown necrotic lesions were observed on mandarin (Citrus reticulata) leaves in Calabria region in southern Italy. The fruiting bodies (acervuli) of the fungus appeared on the necrotic tissues. Isolation was made on potato dextrose agar (PDA) from small sections (3-5 mm2) of lesions margins after surface disinfection in 1% NaOCl. The isolated fungus developed cottony, grey with aerial mycelium with stromatic acervuli producing orange masses of conidia. Conidia were hyaline, cylindrical tapering slightly from only one side measuring 16 - 20 × 3.5 - 5.5 ?m. These morphological criteria matched those of Colletotrichum kahawae clade (Weir et al., 2012). Fungal identity at species level was further confirmed by amplification and sequencing of the beta-tubulin (TUB), partial histone 3 (HIS3) and internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) gene regions as described by Damm et al. (2012). The TUB sequence (GenBank accession No. KU605630) shared 100% homology with the TUB sequence of C. kahawae subsp. ciggaro (KC297082). The sequences of HIS3 (KU605631) and ITS (KU605632) were 100% homologous with KC297048 and GU174550, respectively. The isolated fungus was tested for pathogenicity on detached mandarin leaves. A 5-mm2 mycelial plugs were applied on the wounded leaves and kept for 7 days at 25°C under high relative humidity (RH). Symptoms developed were similar to those observed on the trees. Re- isolation revealed the same morphology of the original isolate. To our knowledge this is the first report of C. kahawae subsp. ciggaro on mandarin in Italy.

In summer of 2014, brown to black necrotic lesions surrounded by yellow haloes were observed on leaves of tomato plants cultivated in Behira governorate. Isolation was made from 3-5 mm2 pieces of lesions margins onto potato dextrose agar (PDA) after surface disinfected using 0.5% NaOCl. The examined morphological features (colony and conidia) were reminiscent of Curvularia spicifera as described by Jeon et al. (2015). The identity was further confirmed by DNA amplification and sequencing of the glyceraldehyde-3-phosphate dehydrogenase (gpd) and second largest subunit of RNA polymerase II (rpb2) gene regions using the same primer sets used in the study of Madrid et al. (2014). Sequences of gpd and rpb2 were deposited in GenBank under accession Nos. KU133371 and KU133372, respectively, and revealed similarity 100% to (KC928089) and 99% to (HF934818) of C. spicifera.Koch's postulates were further confirmed using surface disinfected leaves cv. Super strain-B wounded by sterile needle. Approximately 3-mm2 of colonized PDA plugs of 7-days-old cultures were placed on the wounded sites. Control leaves and fruit were wounded and inoculated with sterile PDA plugs. Tomato leaves were placed at room temperature (28-30°C) and 80% of RH. Five days later, brown circular necrotic lesions (2-3 mm2) were developed on the inoculated leaves. The control leaves showed no symptoms. Re-isolation from infected tissues revealed C. spicifera and its identity was morphologically confirmed. To our knowledge this the first report of C. spicifera (Bainier) Boedijn causing leaf spot on tomato in Egypt.

During the 2009 and the 2010 growing seasons, a root rot disease has been detected on young potted Persea americana plants in two nurseries located in the Catania and Messina provinces (eastern Sicily, Italy). A Cylindrocarpon sp. was consistently recovered from pieces of symptomatic tissues on Petri dishes containing potato dextrose agar. On the basis of morphological characteristics and molecular identification by DNA sequencing and phylogenetic analysis of internal transcribed spacer and ?-tubulin gene regions, the causal agent was identified as Ilyonectria (=Neonectria) macrodidyma. Koch's postulates were fulfilled by pathogenicity tests carried out on potted P. americana seedlings. To our knowledge, this is the first to report worldwide of the occurrence of a disease caused by I. macrodidyma on P. americana.

Novel species of fungi described in the present study include the following from Australia: Neoseptorioideseucalypti gen. & sp. nov. from Eucalyptus radiata leaves, Phytophthora gondwanensis from soil, Diaporthetulliensis from rotted stem ends of Theobroma cacao fruit, Diaporthe vawdreyi from fruit rot of Psidium guajava,Magnaporthiopsis agrostidis from rotted roots of Agrostis stolonifera and Semifissispora natalis from Eucalyptusleaf litter. Furthermore, Neopestalotiopsis egyptiaca is described from Mangifera indica leaves (Egypt), Roussoellamexicana from Coffea arabica leaves (Mexico), Calonectria monticola from soil (Thailand), Hygrocybe jackmaniifrom littoral sand dunes (Canada), Lindgomyces madisonensis from submerged decorticated wood (USA), Neofabraeabrasiliensis from Malus domestica (Brazil), Geastrum diosiae from litter (Argentina), Ganoderma wiiroenseon angiosperms (Ghana), Arthrinium gutiae from the gut of a grasshopper (India), Pyrenochaeta telephoni from thescreen of a mobile phone (India) and Xenoleptographium phialoconidium gen. & sp. nov. on exposed xylem tissuesof Gmelina arborea (Indonesia). Several novelties are introduced from Spain, namely Psathyrella complutensis onloamy soil, Chlorophyllum lusitanicum on nitrified grasslands (incl. Chlorophyllum arizonicum comb. nov.), Aspergilluscitocrescens from cave sediment and Lotinia verna gen. & sp. nov. from muddy soil. Novel foliicolous taxa from SouthAfrica include Phyllosticta carissicola from Carissa macrocarpa, Pseudopyricularia hagahagae from Cyperaceaeand Zeloasperisporium searsiae from Searsia chirindensis. Furthermore, Neophaeococcomyces is introduced asa novel genus, with two new combinations, N. aloes and N. catenatus. Several foliicolous novelties are recordedfrom La Réunion, France, namely Ochroconis pandanicola from Pandanus utilis, Neosulcatispora agaves gen. &sp. nov. from Agave vera-cruz, Pilidium eucalyptorum from Eucalyptus robusta, Strelitziana syzygii from Syzygiumjambos (incl. Strelitzianaceae fam. nov.) and Pseudobeltrania ocoteae from Ocotea obtusata (Beltraniaceae emend.).Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

Novel species of fungi described in this study include those from various countries as follows: Australia, Chaetopsina eucalypti on Eucalyptus leaf litter, Colletotrichum cobbittiense from Cordyline stricta x C. australis hybrid, Cyanodermella banksiae on Banksia ericifolia subsp. macrantha, Discosia macrozamiae on Macrozamia miquelii, Elsinoe banksiigena on Banksia marginata, Elsinoe elaeocarpi on Elaeocarpus sp., Elsinoe leucopogonis on Leucopogon sp., Helminthosporium livistonae on Livistona australis, Idriellomyces eucalypti (incl. Idriellomyces gen. nov.) on Eucalyptus obliqua, Lareunionomyces eucalypti on Eucalyptus sp., Myrotheciomyces corymbiae (incl. Myrotheciomyces gen. nov., Myrotheciomycetaceae fam. nov.), Neolauriomyces eucalypti (incl. Neolauriomyces gen. nov., Neolauriomycetaceae fam. nov.) on Eucalyptus sp., icamyces eucalypti (incl. icamyces gen. nov.) on Eucalyptus leaf litter, Oidiodendron eucalypti on Eucalyptus maidenii, Paracladophialophora cyperacearum (incl. Paracladophialophoraceae fam. nov.) and Periconia cyperacearum on leaves of Cyperaceae, Porodiplodia livistonae (incl. Porodiplodia gen. nov., Porodiplodiaceae fam. nov.) on Livistona australis, Sporidesmium melaleucae (incl. Sporidesmiales ord. nov.) on Melaleuca sp., Teratosphaeria sieberi on Eucalyptus sieberi, Thecaphora australiensis in capsules of a variant of Oxalis exilis. Brazil, Aspergillus serratalhadensis from soil, Diaporthe pseudo-inconspicua from Poincianella pyramidalis, Fomitiporella pertenuis on dead wood, Geastrum magnosporum on soil, Marquesius aquaticus (incl. Marquesius gen. nov.) from submerged decaying twig and leaves of unidentified plant, Mastigosporella pigmentata from leaves of Qualea parviflorae, Mucor souzae from soil, Mycocalia aquaphila on decaying wood from tidal detritus, Preussia citrullina as endophyte from leaves of Citrullus lanatus, Queiroziella brasiliensis (incl. Queiroziella gen. nov.) as epiphytic yeast on leaves of Portea leptantha, Quixadomyces cearensis (incl. Quixadomyces gen. nov.) on decaying bark, Xylophallus clavatus on rotten wood. Canada, Didymella cari on Carum carvi and Coriandrum sativum. Chile, Araucasphaeria foliorum (incl. Araucasphaeria gen. nov.) on Araucaria araucana, Aspergillus tumidus from soil, Lomentospora valparaisensis from soil. Colombia, Corynespora pseudocassiicola on Byrsonima sp., Eucalyptostroma eucalyptorum on Eucalyptus pellita, Neometulocladosporiella eucalypti (incl. Neometulocladosporiella gen. nov.) on Eucalyptus grandis x urophylla, Tracylla eucalypti (incl. Tracyllaceae fam. nov., Tracyllalales ord. nov.) on Eucalyptus urophylla. Cyprus, Gyromitra anthracobia (incl. Gyromitra subg. Pseudoverpa) on burned soil. Czech Republic, Lecanicillium restrictum from the surface of the wooden barrel, Lecanicillium testudineum from scales of Trachemys scripta elegans. Ecuador, Entoloma yanacolor and Saproamanita quitensis on soil. France, Lentithecium carbonneanum from submerged decorticated

Novel species of fungi described in this study include those from various countries as follows: Angola, Gnomoniopsis angolensis and Pseudopithomyces angolensis on unknown host plants. Australia, Dothiora corym­biae on Corymbia citriodora, Neoeucasphaeria eucalypti (incl. Neoeucasphaeria gen. nov.) on Eucalyptus sp., Fumagopsis stellae on Eucalyptus sp., Fusculina eucalyptorum (incl. Fusculinaceae fam. nov.) on Eucalyptus socialis, Harknessia corymbiicola on Corymbia maculata, Neocelosporium eucalypti (incl. Neocelosporium gen. nov., Neocelosporiaceae fam. nov. and Neocelosporiales ord. nov.) on Eucalyptus cyanophylla, Neophaeomoniella corymbiae on Corymbia citriodora, Neophaeomoniella eucalyptigena on Eucalyptus pilularis, Pseudoplagiostoma corymbiicola on Corymbia citriodora, Teratosphaeria gracilis on Eucalyptus gracilis, Zasmidium corymbiae on Corymbia citriodora. Brazil, Calonectria hemileiae on pustules of Hemileia vastatrix formed on leaves of Coffea arabica, Calvatia caatinguensis on soil, Cercospora solani-betacei on Solanum betaceum, Clathrus natalensis on soil, Diaporthe poincianellae on Poincianella pyramidalis, Geastrum piquiriunense on soil, Geosmithia carolliae on wing of Carollia perspicillata, Henningsia resupinata on wood, Penicillium guaibinense from soil, Periconia caespitosa from leaf litter, Pseudocercospora styracina on Styrax sp., Simplicillium filiforme as endophyte from Citrullus lanatus, Thozetella pindobacuensis on leaf litter, Xenosonderhenia coussapoae on Coussapoa floccosa. Canary Islands (Spain), Orbilia amarilla on Euphorbia canariensis. Cape Verde Islands, Xylodon jacobaeus on Eucalyptus camaldulensis. Chile, Colletotrichum arboricola on Fuchsia magellanica. Costa Rica, Lasiosphaeria miniovina on tree branch. Ecuador, Ganoderma chocoense on tree trunk. France, Neofitzroyomyces nerii (incl. Neofitzroyomyces gen. nov.) on Nerium oleander. Ghana, Castanediella tereticornis on Eucalyptus tereticornis, Falcocladium africanum on Eucalyptus brassiana, Rachicladosporium corymbiae on Corymbia citriodora. Hungary, Entoloma silvae-frondosae in Carpinus betulus-Pinus sylvestris mixed forest. Iran, Pseudopyricularia persiana on Cyperus sp. Italy, Inocybe roseascens on soil in mixed forest. Laos, Ophiocordyceps houaynhangensis on Coleoptera larva. Malaysia, Monilochaetes melastomae on Melastoma sp. Mexico, Absidia terrestris from soil. Netherlands, Acaulium pannemaniae, Conioscypha boutwelliae, Fusicolla septimanifiniscientiae, Gibellulopsis simonii, Lasionectria hilhorstii, Lectera nordwiniana, Leptodiscella rintelii, Parasarocladium debruynii and Sarocladium dejongiae (incl. Sarocladiaceae fam. nov.) from soil. New Zealand, Gnomoniopsis rosae on Rosa sp. and Neodevriesia metrosideri on Metrosideros sp. Puerto Rico, Neodevriesia coccolobae on Coccoloba uvifera, Neodevriesia tabebuiae and Alfaria tabebuiae on Tabebuia chrysantha. Russia, Amanita paludosa on bogged soil in mixed deciduous forest, Entoloma tiliae in forest of Til

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.

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium species and is a common contaminant of a wide variety of food commodities, with Aspergillus carbonarius being the main producer of OTA contamination in grapes and wine. The molecular structure of OTA comprises a dihydroisocoumarin ring linked to phenylalanine and, as shown in different producing fungal species, a polyketide synthase (PKS) is a component of the OTA biosynthetic pathway. Similar to observations in other filamentous ascomycetes, the genome sequence of A. carbonarius contains a large number of genes predicted to encode PKSs. In this work a pks gene identified within the putative OTA cluster of A. carbonarius, designated as AcOTApks, was inactivated and the resulting mutant strain was unable to produce OTA, confirming the role of AcOTApks in this biosynthetic pathway. AcOTApks protein is characteristic of the highly reduced (HR)-PKS family, and also contains a putative methyltransferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. AcOTApks is different from the ACpks protein that we previously described in A. carbonarius, which showed an expression profile compatible with OTA production. We performed phylogenetic analyses of the beta-ketosynthase and acyl-transferase domains of the OTA PKSs that had been identified and characterized in different OTA producing fungal species. The phylogenetic results were similar for both domains analyzed and showed that OTA PKS of A. carbonarius, Aspergillus niger and Aspergillus ochraceus clustered in a monophyletic group with 100% bootstrap support suggesting a common origin, while the other OTA PKSs analyzed were phylogenetically distant. A quantitative RT-PCR assay monitored AcOTApks expression during fungal growth and concomitant production of OTA by A. carbonarius in synthetic grape medium. A clear correlation between the expression profile of AcOTApks and kinetics of OTA production was observed, with AcOTApks reaching its maximum level of transcription before OTA accumulation in mycelium reached its highest level, confirming the fact that gene transcription always precedes phenotypic production. (C) 2014 Elsevier B.V. All rights reserved.

Penicillium is a diverse genus occurring worldwide and its species play important roles as decomposers of organic materials and cause destructive rots in the food industry where they produce a wide range of mycotoxins. Other species are considered enzyme factories or are common indoor air allergens. Although DNA sequences are essential for robust identification of Penicillium species, there is currently no comprehensive, verified reference database for the genus. To coincide with the move to one fungus one name in the International Code of Nomenclature for algae, fungi and plants, the generic concept of Penicillium was re-defined to accommodate species from other genera, such as Chromocleista, Eladia, Eupenicillium, Torulomyces and Thysanophora, which together comprise a large monophyletic clade. As a result of this, and the many new species described in recent years, it was necessary to update the list of accepted species in Penicillium. The genus currently contains 354 accepted species, including new combinations for Aspergillus crystallinus, A. malodoratus and A. paradoxus, which belong to Penicillium section Paradoxa. To add to the taxonomic value of the list, we also provide information on each accepted species MycoBank number, living ex-type strains and provide GenBank accession numbers to ITS, ?-tubulin, calmodulin and RPB2 sequences, thereby supplying a verified set of sequences for each species of the genus. In addition to the nomenclatural list, we recommend a standard working method for species descriptions and identifications to be adopted by laboratories working on this genus.

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.

Hydroxycinnamic acids (HCAs), phenolic components of wine, are known to have antimicrobial properties. Aspergillus carbonarius is one of the most important ochratoxin A (OTA) producing fungi in wine. Strategies for the control and prevention of A. carbonarius contamination are important for the maintenance of wine safety. This study sought to determine the potential of HCAs, such as caffeic, p-coumaric and ferulic acids, as antifungal natural compounds for the control of A. carbonarius growth and OTA production. The HCAs were tested at the increasing concentrations of 0.30, 0.65 and 1.10 mg/ml in minimal medium (MM) and grape juice. Germination of conidia was not affected in neither of the two media in presence of HCAs. At all the concentrations tested, OTA biosynthesis in MM was reduced and the dose effect was more evident for p-coumaric and ferulic acids; in grape juice the reduction trend was confirmed, and ferulic acid showed the highest inhibitory effect. Moreover, the expression level of genes encoding a polyketide synthase (AcOTApks) and a nonribosomal peptide synthetase (AcOTAnrps) involved in OTA biosynthesis, was evaluated by real-time PCR in A. carbonarius grown in presence of 0.65 mg/ml of HCAs. From gene expression analysis only the AcOTApks gene showed a marked reduction of transcription level in presence of p-coumaric and ferulic acids. On the contrary, caffeic acid seemed to not influence the expression levels of the genes analysed in this study, suggesting a different mechanism of action on the regulation of OTA biosynthesis.

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.

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/).

Almonds are extremely nutrient-dense nuts. They provide generous amounts of calories, fats, complex carbohydrates, protein, vitamins, minerals and fiber. They are consumed as raw nuts or as ingredients of derived products. However, almonds can be susceptible to aflatoxins contamination, extremely harmful for the human health even at low concentrations. We evaluated the evolution of aflatoxins during processing of almonds into traditional Italian products, such as pastries, nougat and syrup. The mass balance approach was used to determine levels and distribution of aflatoxins in each fraction collected during processing steps. Experiments were conducted on almonds inoculated with a toxigenic strain of Aspergillus flavus. A robust and horizontal HPLC-FLD method was optimized and used for aflatoxins determination in all the matrices considered in this study. The two tested blanching processes (steaming and boiling in water) did not reduce aflatoxins levels in peeled almonds. Standard roasting conditions produced up to 50% reduction of aflatoxins. Higher aflatoxin reduction (82%) was observed by roasting at 150°C for 120 min, but almonds lost their organoleptic characteristics. The preparation and cooking of nougat produced a consistent reduction of 54-70% of aflatoxins due to the caramelisation of sugar on almond surface. Almond pastries were prepared by mixing almond paste, eggs and sugar and cooked at 140°C for 30 min, 160°C for 20 min or 180°C for 15 min. Aflatoxins were substantially stable during cooking of pastries. Almond syrup was prepared from ground peeled almond that was infused in water for 5 hours. After infusion spent almonds were discarded and the infuse was sugared and boiled until it reaches the consistency of a syrup. About 18-25% of total aflatoxins passed in the final syrup. The whole process of almond syrup preparation produced a marked increase of total aflatoxins. This increase was probably due to the activation of endogenous almond enzymes that release free aflatoxins from modified aflatoxins during the water infusion of ground peeled almonds.

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

The Microbial Resource Research Infrastructure (MIRRI) (http://www.mirri.org/) has been launched in 2010 within the Biomedical Research Infrastructures of the European Strategy Forum on Research Infrastructures (ESFRI) initiative.The current configuration of MIRRI involves several countries (Portugal, Spain, France, Greece, Belgium, Poland and Latvia have already signed a Memorandum of Understanding), which are called to support the implementation of the Central Coordination Unit (CCU), their National Nodes (NN) and the needs of the national communities. While the CCU is clearly defined at the European level by an agreement among interested countries, the NN may follow various forms.In Italy there are numerous collections of microorganisms disseminated in the country, which can be exploited to boost technological innovations and to face different societal challenges. Indeed, these collections are often not managed according to international standards and their catalogues are not online displayed. Moreover, coordination among these collections is still limited. In this contest, the creation of an effective Italian network of microbial culture collections can be fundamental to implement the recently presented national bioeconomy strategy and to support the needs of the companies acting in different sectors, as well as to foster the signature of the Memorandum of Understanding of MIRRI, which has not yet been done by the relevant National Agency.For these reasons, a Joint Research Unit for the implementation of the Italian node of MIRRI (MIRRI-IT JRU) (http://www.mirri-it.it) has recently been founded with the contribution of the Universities of Turin, Perugia, Modena and Reggio Emilia, the University Hospital San Martino Genoa and the Italian National Research Council. The main goal of the MIRRI-IT JRU is the development of a tight network of Italian collections of microbial resources. The mission is to overcome fragmentation in availability of resources and services, enhancing the quality management system of the collections while focusing on needs and challenges of the stakeholders interested in the biotechnological transfer of these resources. Therefore, the main activities of the MIRRI-IT JRU are meant to:a.coordinate and support the operation of the Microbial Culture Collections in Italy according to the established international quality standards;b.harmonize the procedures of the Collections in order to comply with national and international rules (i.e. Nagoya Protocol, Intellectual Property Rights, privacy, biosafety, etc.);c.propose actions to relevant State authorities in order to fortify the functioning and sustainability of the Collections;d.promote interdisciplinary cooperation and represent Italy in relevant national and international Networks and Organizations;e.provide an unique entry point to quality microbiological services and microbial Biological Resource Centres (mBRC) holdings.

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.

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.

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

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

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 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.

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

Fermented meat products are unique and often represented as an element of culinary heritage and gastronomic identity. Together with meat enzymes and bacteria, molds are very important in the ripening of some dry fermented meat products. Fungal starter cultures, contribute to the development of the typical sausage's flavor through their lipolytic and proteolytic activities. They also played an important role in preventing lipid oxidations and counteracting undesirable microorganisms. Various genera of fungi could colonize salami, recently we described a new promising species P. salamii, and its technological features. In fact, our data evidencing its interesting attitude to the seasoning process of meat resulted in fast growth, with high lipolytic and proteolytic activities suggesting it as promising candidate to be used in new fungal starter formulations for meat industry. On the other hand, depending on its peculiar composition, the surface mycobiota could be colonized by undesirable molds, like P. nordicum an important and consistent producer of the potent nephrotoxin ochratoxin A (OTA), widely reported as contaminant of dry-cured meat products. We developed a sensitive and easy to use LAMP assay for P. nordicum detection on salami surface co-inoculated with P. nalgiovense and P. nordicum at different rates. In addition, we monitored the expression of a keygene of OTA biosynthesis in P. nordicum and toxin accumulation in meat during the seasoning process, observing that expression profile was consistent with OTA accumulation. Results revealed that P. nordicum monitoring, since early steps of seasoning, could represent a valid and fast molecular tool for early alert of the possible OTA accumulation.

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.

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

In the nineties fungal secondary metabolites (SMs), such as antibiotics and mycotoxins, started to be genetically characterized. Then, the clustered arrangement of genes involved in the biosynthesis of a single SM was studied. In the pre-genomic era, gene cluster discovery in fungi was complex and time-consuming, involving cumbersome traditional molecular methods. Genomics has revolutionized the research on SM biosynthesis pathways, allowing the bypass of such approaches. The breakthrough of next-generation sequencing (NGS) technologies and the advent of Bioinformatics have opened a new era in the study of biological systems. NGS technologies contributed significantly to the increasing availability of fungal genomes and bioinformatic analysis lead to the identification of SM clusters of known metabolites and to the prediction of novel cryptic clusters for still unknown microbial metabolites. However, most of the clusters identified by genome analysis are still to be deeply examined to completely understand the pathway steps and the regulatory network behind the metabolite biosynthesis. Here, we present the example of how the genomic approach has led to the identification of biosynthetic genes and their role in ochratoxin A (OTA) production by Aspergillus carbonarius. From the genome sequencing and the subsequent prediction of OTA cluster, we demonstrated by gene knock-out approach the key role of three genes (AcOTApks, AcOTAnrps and AcOTAhal) in the OTA biosynthesis. Single gene knock-out mutant allowed us to elucidate the order of the enzymatic steps in the biosynthesis pathway. Other predicted genes in the cluster, such as a p450 monooxygenase and a transcription factor gene, need to be investigated for the full knowledge of the structural and regulatory mechanisms of toxin production. Furthermore, transcriptomic analyses are in progress to study and clarify at a deeper level the complex genetic picture of the fungus during OTA biosynthesis.

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

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.

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

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

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.

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.

Polyketide synthase (PKSs) and nonribosomal peptide synthetase (NRPSs) arelarge multimodular enzymes involved in biosynthesis of polyketide and peptide toxinsproduced by fungi. Furthermore, hybrid enzymes, in which a reducing PKS region is fusedto a single NRPS module, are also responsible of the synthesis of peptide-polyketidemetabolites in fungi. The genes encoding for PKSs and NRPSs have been exposed tocomplex evolutionary mechanisms, which have determined the great number and diversityof metabolites. In this study, we considered the most important polyketide and peptidemycotoxins and, for the first time, a phylogenetic analysis of both PKSs and NRPSsinvolved in their biosynthesis was assessed using two domains for each enzyme:?-ketosynthase (KS) and acyl-transferase (AT) for PKSs; adenylation (A) andcondensation (C) for NRPSs. The analysis of both KS and AT domains confirmed thedifferentiation of the three classes of highly, partially and non-reducing PKSs. HybridPKS-NRPSs involved in mycotoxins biosynthesis grouped together in the phylogenetictrees of all the domains analyzed. For most mycotoxins, the corresponding biosyntheticenzymes from distinct fungal species grouped together, except for PKS and NRPSinvolved in ochratoxin A biosynthesis, for which an unlike process of evolution could behypothesized in different species.

Aspergillus comprises a diverse group of species based on morphological, physiological and phylogenetic characters, which significantly impact biotechnology, food production, indoor environments and human health. Aspergillus was traditionally associated with nine teleomorph genera, but phylogenetic data suggest that together with genera such as Polypaecilum, Phialosimplex, Dichotomomyces and Cristaspora, Aspergillus forms a monophyletic clade closely related to Penicillium. Changes in the International Code of Nomenclature for algae, fungi and plants resulted in the move to one name per species, meaning that a decision had to be made whether to keep Aspergillus as one big genus or to split it into several smaller genera. The International Commission of Penicillium and Aspergillus decided to keep Aspergillus instead of using smaller genera. In this paper, we present the arguments for this decision. We introduce new combinations for accepted species presently lacking an Aspergillus name and provide an updated accepted species list for the genus, now containing 339 species. To add to the scientific value of the list, we include information about living ex-type culture collection numbers and GenBank accession numbers for available representative ITS, calmodulin, ?-tubulin and RPB2 sequences. In addition, we recommend a standard working technique for Aspergillus and propose calmodulin as a secondary identification marker.

Members of the economically important ascomycete genus Trichoderma are ubiquitously distributed around the world. The mycoparasitic lifestyle and plant defence-inducing interactions of Trichoderma spp. make them ideal biocontrol agents. Of the Trichoderma enzymes that produce secondary metabolites, some of which likely play important roles in biocontrol processes, polyketide synthase (PKSs) have garnered less attention than non-ribosomal peptide synthetases such as those that produce peptaibols. We have taken a phylogenomic approach to study the PKS repertoire encoded in the genomes of Trichoderma reesei, Trichoderma atroviride and Trichoderma virens. Our analysis lays a foundation for future research related to PKSs within the genus Trichoderma and in other filamentous fungi.

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.

Objectives. Almonds and apricot kernels have several health benefits. They are a good source of proteins, fiber, unsaturated fats, vitamins and minerals. They are consumed as raw nuts or as ingredients of derived products. However they can be contaminated by aflatoxins (AFs), well known carcinogenic mycotoxins. We evaluated the evolution of AFs during transformation processes of almonds into traditional Italian products, such as pastries, nougat and syrup which involves blanching, peeling, cooking, roasting and water infusion. The efficacy of sorting to segregate AFs contaminated apricot kernels was also assessed. Materials and MethodsExperiments were conducted on naturally contaminated apricot kernels and almonds inoculated with a toxigenic strain of Aspergillus flavus. A robust and horizontal HPLC-FLD method was optimized and used for AFs determination in all the matrices considered in this study1.ResultsBlanching processes (by steaming or boiling in water) did not reduce AFs levels in peeled almonds and apricot kernels. Standard roasting conditions produced up to 50% reduction of AFs. During nougat preparation it was observed a 54-70% AFs reduction due to the caramelisation of sugar on almond surface. AFs were instead stable during cooking of pastries. During almond syrup preparation 18-25% of AFs passed in the final syrup and the whole process produced a marked increase of total AFs. This increase was probably due to the water infusion of ground peeled almonds that activates endogenous almond enzymes that release free AFs from masked AFs1. To reduce AFs contamination in apricot kernels we applied the manual colour sorting to peeled kernels and obtained excellent results: 97-99% AFs reduction by removing only discoloured kernels.ConclusionsRoasting, caramelisation and manual sorting of peeled nuts were identified as effective processes to reduce AFs in nuts. We observed, for the first time, a marked increase of AFs during almond syrup preparation which suggests the existence of masked AFs never reported before. The separation of discolored apricot kernels is a feasible strategy to remove almost all AFs in contaminated peeled kernels. These information are extremely useful and can be exploited by food producers to improve the safety of nuts and derived products.1 Zivoli et al. 2014. Effect of Almond Processing on Levels and Distribution of Aflatoxins in Finished Products and By-products. J. Agric. Food Chem. 62: 5707-5715.

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

Recently, studies on the molecular aspects of ochratoxin A biosynthesis have significantly advanced. Differently from other mycotoxins, the genes and the enzymatic stages involved in biosynthesis pathway of ochratoxin A have remained long unknown. New ecological data have led to the definition of new producing species, responsible of ochratoxin A contamination in several food and feed. In parallel, genomics, transcriptomics and proteomics studies have provided new information to better define the molecular key steps of the mycotoxin biosynthesis. Further studies are still needed to completely clarify the regulatory mechanisms underlying the activation of the production of ochratoxin A. Previous findings on fungal secondary metabolism biosynthesis and the availability of new data from different omics approaches will permit to fill the gap of knowledge in the near future.

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.

Aspergillus is a diverse fungal genus containing many species of great agricultural, biotechnological and medical relevance. Because of the broad use of the genus name in diverse disciplines, and the importance of individual species names in these areas, the taxonomy and nomenclature of Aspergillus should remain stable. A formal proposal to change the generic type from A. glaucus to A. niger was recently published. Here we present arguments against this proposal. We assert that it should be rejected because it will not ensure nomenclatural stability for Aspergillus, and will put the names of several important species, such as A. flavus, A. fumigatus and A. oryzae at risk of being classified in different genera and being lost.

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.

Mycotoxins are major food contaminants affecting global food security, especially in low and middle-income countries. The European Union (EU) funded project, MycoKey, focuses on "Integrated and innovative key actions for mycotoxin management in the food and feed chains" and the right to safe food through mycotoxin management strategies and regulation, which are fundamental to minimizing the unequal access to safe and sufficient food worldwide. As part of the MycoKey project, a Mycotoxin Charter (charter.mycokey.eu) was launched to share the need for global harmonization of mycotoxin legislation and policies and to minimize human and animal exposure worldwide, with particular attention to less developed countries that lack effective legislation. This document is in response to a demand that has built through previous European Framework Projects--MycoGlobe and MycoRed--in the previous decade to control and reduce mycotoxin contamination worldwide. All suppliers, participants and beneficiaries of the food supply chain, for example, farmers, consumers, stakeholders, researchers, members of civil society and government and so forth, are invited to sign this charter and to support this initiative.

Ochratoxin A (OTA) is a potent pentaketide nephrotoxin diffusely distributed in food and feed products (grains, legumes, coffee, dried fruits, meats, beer and wine); it is also carcinogenic, neurotoxic, teratogenic and immunotoxic. This mycotoxin is produced by species of genus Aspergillus and Penicillium. OTA is the primarily mycotoxin risk in wine and dried vine fruits. Several studies focused on Aspergillus section Nigri, due to their role as causative agents of black rot of grapes, and subsequently as cause of ochratoxin A contamination. Nine different black Aspergillus species have been identified on grapes with different secondary metabolites profiles. These species are often difficult to be identified by means of classical methods. The polyphasic approach used in our studies led to characterization of three new non toxigenic species occurring on grapes: A. brasiliensis, A. ibericus and A. uvarum. However, the main source of OTA contamination in grapes is A. carbonarius, followed by A. niger and A. welwitschiae. This contamination is strongly related to climatic conditions, geographical regions (South Mediterranean climate is highly conducive), grape varieties, damage by insects, growing season (high susceptibility from early veraison to harvest, with a peak at ripening), and great variations may occur from one year to another. Differently from other mycotoxins, the genes and the enzymatic stages involved in OTA biosynthesis pathway have remained unknown for long. In last years, genomics, transcriptomics and proteomics studies have provided new information to better define the molecular key steps of OTA biosynthesis. Genome sequencing of A. carbonarius led us to predict OTA cluster and to elucidate the key role of three genes (AcOTApks, AcOTAnrps and AcOTAhal) and the order of the enzymatic steps of the biosynthesis pathway. Other predicted genes in the cluster have been identified and analysed, such as a p450 monooxygenase and a transcription factor gene, likely involved in the structural and regulatory mechanisms of OTA production. Furthermore, transcriptomic analyses are in progress to study and clarify the complex genetic picture of the fungus during OTA biosynthesis at a deeper level. Interestingly, recent studies on climate change effects evidenced the influence of raising temperature and CO2 levels on OTA production increase. Managing OTA contamination to reduce risks in grapes implies several strategies, such as implementations of good agricultural practices and risk maps, in association with the use of insecticides and fungicides when favourable climatic conditions occur. In addition, corrective actions can be adopted in wineries.

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

The genus Aspergillus is among the most abundant and widely distributed organisms on earth, and comprises approximately 350 accepted species. Economically it is one of the most important fungal genera for biotechnological and industrial use (enzymes, organic acids, active metabolites), but members of the genus are also frequently reported as foodborne contaminants (food spoilage and mycotoxin contamination), or as causative agents of human mycoses (pulmonary aspergillosis, otomycosis, keratitis). Recently, the ICN adopted the single name nomenclature which has forced mycologists to choose one name for fungi (i.e Aspergillus, Fusarium, Penicillium, etc.). In this respect, the phylogenetic approach was mainly used to settle the disputes on the right choice with decisions not affecting the majority of the concerned fungal group. This is not the case for the genus Aspergillus, because it is characterized by a well-defined asexual fruiting structure, but is very broad in concept, as it is associated with eleven sections with a sexual state. Two proposals for the single name nomenclature in Aspergillus are presented: one attributes the name "Aspergillus" to clades comprising seven different teleomorphic names, by supporting the monophyly of this genus. The other proposes that Aspergillus is a non-monophyletic genus, by preserving the Aspergillus name only to species belonging to subgenus Circumdati and maintaining the sexual names in the other lades. The aim of our study was to test the monophyly of Aspergilli by a multilocus phylogenetic approach which was applied by two independent analyses. One was run on the publicly available coding regions of six genes (RPB1, RPB2, Tsr1, Cct8, BenA, CaM), on 96 species of Penicillium, Aspergillus and related taxa. Bayesian and Ultrafast Maximum Likelihood (IQ-Tree) and RaxML analyses gave the same conclusion with highly supporting the monophyly of Aspergillus. The other analyses were also made by using publicly available data by using the coding sequences of nine loci (18S rRNA, 5,8S rRNA, 28S rRNA (D1-D2), RPB1, RPB2, CaM, BenA, Tsr1, Cct8) of 150 different species. Both Bayesian (MrBayes) and Maximum Likelihood (RAxML) trees obtained by this second round of independent analyses strongly supported the monophyly of the genus Aspergillus. The stability test also confirmed the robustness of the results obtained. In conclusion both conducted statistical analyses reject the hypothesis that Aspergilli are non-monophyletic, and gives robust arguments that the genus is monophyletic and clearly separated from the monophyletic genus Penicillium. We therefore conclude that there is no phylogenetic evidence to split Aspergillus and the name Aspergillus can be used for all the species belonging to Aspergillus i.e. the clade comprising the subgenera Aspergillus, Circumdati, Fumigati, Nidulantes, section Cremei and certain species which were formerly part of the genera Phialosimplex and Polypaecilum.