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.

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

The human respiratory tract represents the major portal of entry for numerous microorganisms, primarily those occurring as airborne particles such as viral and bacterial entities, or fungal spores. Microorganism characteristics coupled with the local host immune response will determine whether they will be cleared or adhere and colonize the airways leading to acute or chronic pulmonary disease. Like bacteria, fungi can cause severe lung diseases, but their infection rates are much lower. The lung microbiota is commonly sampled using relatively invasive bronchoscopic procedures. Exhaled breath condensate (EBC) collection offers a potentially less invasive alternative for lung microbiota sampling. This study tries to determine the composition of fungal communities in a cohort of healthy adult volunteer subjects from Puglia (Apulia), Italy.MethodsFungi diversity in 27 EBC samples collected from Italian adult volunteers was investigated using conventional microbiological culturing and DNA sequencing approach.ResultsTen tested subjects (37,03%) turned out to present fungi in the EBC. We observed complex fungal communities, in which more than 10% of the isolated species are represented by Aspergillus sydowii (14,8%) and Cladosporium spp (11,11%). Three subjects that showed fungal presence in EBC have been diagnosed with a respiratory disease.ConclusionsWe present a survey of an important scientific field in its early stages that is fungal contamination of airways of healthy subjects in a small geographic area. Furthermore, we interpreted our results to highlight the potential role of fungi in the context of respiratory diseases.

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

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

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

Fumonisins are a family of carcinogenic secondarymetabolites produced by members of the Fusariumfujikuroi species complex (FFSC) and rare strains ofFusarium oxysporum. In Fusarium, fumonisin biosyntheticgenes (FUM) are clustered, and the cluster isuniform in gene organization. Here, sequence analysesindicated that the cluster exists in five differentgenomic contexts, defining five cluster types. In FUMgene genealogies, evolutionary relationships betweenfusaria with different cluster types were largely incongruentwith species relationships inferred fromprimary-metabolism (PM) gene genealogies, and FUMcluster types are not trans-specific. In addition, synonymoussite divergence analyses indicated that threeFUM cluster types predate diversification of FFSC. Thedata are not consistent with balancing selection orinterspecific hybridization, but they are consistentwith two competing hypotheses: (i) multiple horizontaltransfers of the cluster from unknown donors to FFSCrecipients and (ii) cluster duplication and loss (birthand death). Furthermore, low levels of FUM gene divergencein F. bulbicola, an FFSC species, and F. oxysporumprovide evidence for horizontal transfer of thecluster from the former, or a closely related species, tothe latter. Thus, uniform gene organization within theFUM cluster belies a complex evolutionary history thathas not always paralleled the evolution of Fusarium.

Blue mold is one of the most important postharvest diseases of pome fruit in all producing countries. Its causal agent, Penicillium expansum, is also known to produce the mycotoxin patulin, with mutagenic, immunotoxic, and neurotoxic properties. Aims of the present study were to identify Penicillium isolates associated with blue mould decay of pome fruits in Apulia region (South Italy), verify if their genetic potential to produce patulin corresponded to actual toxin contamination, and compare their in vitro and in vivo toxigenicity. Twenty-nine isolates of Penicillium spp. were recovered from apples and pears with blue mold symptoms. Fruits were analyzed for patulin content and results were compared with in vitro toxin production. In general, patulin production was more conspicuous in vivo (particularly on Golden Delicious apples) than in vitro, although the stronger in vivo producer did not correspond to the stronger in vitro producer. Isolate identification was based on both morphological characters and DNA analysis by PCR amplification with P. expansum species-specific primers and sequencing of beta-tubulin gene. Furthermore, fungal isolates were tested for the occurrence of gene (patN) coding the enzyme isoepoxydon dehydrogenase (IDH), involved in the patulin metabolic pathway and considered an useful indicator of critical control points for patulin contamination. All 25 isolates identified as P. expansum were patN and patulin production positive. Moreover, 4 pear isolates belonging to other Penicillium spp. were found, whose identification is being confirmed. They were positive for the patN gene, but only two actually produced patulin. It can be concluded that blue mold of pome fruits in Apulia is mainly associated with toxigenic P. expansum isolates, thus a rapid detection is important to avoid patulin contamination beyond the regulatory limits. Nevertheless, it seems that patN gene alone cannot be considered a predictive assay for production of patulin. An evaluation of its expression level should be carried out.

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

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.

Collectively, species of Fusarium produce a diversity of mycotoxins and other secondary metabolites (SMs), but individual species contribute to only a fraction of this diversity. Research over the past two decades has revealed that in Fusarium and other fungi genes responsible for the biosynthesis of an SM are typically clustered. We are using comparative genomic and phylogenetic analyses to investigate the distribution and evolution of SM biosynthetic gene clusters among Fusarium species. These analyses indicate that the distribution of SM clusters differs markedly among species. For example, the fusarubin gene cluster is present in all species of Fusarium examined to date, while the fusarin cluster is widely but not uniformly distributed among species. The trichothecene and fusaric acid clusters have more limited distributions, but their presence is more uniform within the multispecies lineages in which they occur. The fumonisin and zearalenone clusters also exhibit relatively narrow distributions, but their presence within lineages is highly discontinuous (i.e. presence/absence of the clusters can differ among closely related species). In most cases examined, the presence of a biosynthetic gene cluster in a species is the result of vertical inheritance from ancestral species. In some cases, however, the presence of a cluster, and therefore the ability to produce the corresponding SM, is the result of horizontal transfer from another Fusarium species. Cluster loss appears to be the major contributor to discontinuous distribution of gene clusters within lineages of Fusarium. The fumonisin cluster is exemplary of this phenomenon; we have evidence for intact fumonisin cluster homologs in nine fusaria in the F. fujikuroi species complex, but evidence for no cluster or the cluster in various stages of decay in over 40 other members of this complex. Together, variation in the distribution of SM clusters among fusaria, phylogenetic relationships of cluster homologs, and the presence of some cluster homologs in other fungal genera suggest variability in the origins of clusters. Some clusters were likely introduced into Fusarium via vertical inheritance from an ancestor while others were likely introduced via horizontal transfer.

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

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

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

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.

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

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

Cave cheese is a surface mold-ripened variety of cheese produced also in South of Italy, exploiting fungal population naturally occurring on cave walls, as part of secondary microbiota for ripening. In this study, 148 fungal strains were isolated from 22 independent cave cheese samples, collected in 13 Italian geographical locations, mostly in Apulian area. DNA-based identification showed the presence of twenty-four fungal species in the outer part of the cheese ripened in caves. Aspergillus westerdijkiae and Penicillium biforme resulted the most frequently isolated species, followed by Penicillium roqueforti and Penicillium solitum. The 86% of cheese sample presented at least one toxigenic species and the 45% revealed the presence of ochratoxigenic species, A. westerdijkiae and A. steynii, suggesting possible mycotoxin risk during ripening stage in caves, confirmed by the presence of ochratoxin A (OTA) in the rind of 36% of samples. In conclusion, cave cheese is a susceptible product for toxigenic mold growth and in particular OTA contamination, therefore adeguate scientific tools for matching organolectic consumer expectations and complete safety of food should be developed, as well as spontaneously molded and not monitored cheeses should not be consumed to avoid mycotoxin risk.

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

Fusarium proliferatum is a member of the Fusarium fujikuroi species complex (FFSC) involved in the maize ear rot together with Fusarium verticillioides, which is a very closely related species. Recently, different studies have detected natural fumonisin contamination in wheat kernels and most of them have shown that the main species isolated was F. proliferatum. Fusarium strains obtained from freshly harvested durum wheat samples (2008 to 2011 harvest seasons) from Argentina were characterized through a phylogenetic analysis based on translation elongation factor-1 alpha (EF-1?) and calmodulin (CaM) genes, determination of mating type alleles, and evaluation of fumonisin production capability. The strains were identified as F. proliferatum (72%), F. verticillioides (24%) and other Fusarium species. The ratio of mating type alleles (MAT-1 and MAT-2) obtained for both main populations suggests possible occurrence of sexual reproduction in the wheat fields, although this seems more frequent in F. proliferatum. Phylogenetic analysis revealed greater nucleotide variability in F. proliferatum strains than in F. verticillioides, however this was not related to origin, host or harvest year. The fumonisin-producing ability was detected in 92% of the strains isolated from durum wheat grains. These results indicate that F. proliferatum and F. verticillioides, among the fumonisin producing species, frequently contaminate durum wheat grains in Argentina, presenting a high risk for human and animal health.

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

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

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

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

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/).

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

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

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

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

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

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

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.

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

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

Blue mould is one of the most important postharvest diseases of pome fruit in all producing countries. It is mainly associated to Penicillium expansum that produces the mycotoxin patulin, although other species might be involved. The aim of the present study was to characterise Penicillium isolates associated with blue mould decay of pome fruit marketed in Apulia region (southern Italy), and verify their ability to produce patulin in vitro. Twenty-nine isolates of Penicillium spp. were recovered from pome fruit showing visible blue mould symptoms, and analysed for patulin production. After fungal isolation, the fruits were singularly analysed for patulin content. In general, the isolates proved to produce patulin and most of the pome fruit contained significant amounts of patulin, but there was no quantitative correspondence between in vitro and in vivo toxin accumulation. Isolate identification at species level was based on DNA analysis by P. expansum species-specific primers and sequencing of ?-tubulin gene. Furthermore, fungal isolates were tested for the occurrence of the patN gene coding the enzyme isoepoxydon dehydrogenase (IDH), involved in patulin metabolic pathway and considered a useful indicator of critical control points for patulin contamination. All 26 isolates identified as P. expansum were positive for patN and produced patulin. Moreover, three pear isolates belonging to other Penicillium species were found. They were positive for patN, but only two actually produced patulin. It can be concluded that toxigenic P. expansum isolates are associated with blue mould of pome fruit marketed in Apulia, thus a rapid detection is important to avoid patulin contamination beyond regulatory limits. Nevertheless, the presence of patN gene alone cannot be considered a predictive assay for patulin production. An evaluation of its expression level should be carried out.

Several species of the genus Penicillium were isolated during a survey of the mycobiota of Apulian cave cheesesripened in a cave in Gravina di Puglia, Italy. A novel species, Penicillium gravinicasei, is described in Penicilliumsection Cinnamopurpurea. Its taxonomic novelty was determined using a polyphasic approach, combining phenotypic,molecular (?-tubulin, calmodulin, ITS and DNA dependent RNA polymerase) DNA sequences andmycotoxin production data. Phylogenetic analyses of the RPB2 data showed that isolates of the novel speciesform a clade most closely related to Penicillium cinnamopurpureum and P. parvulum with high bootstrap support.The fungus did not produce ochratoxin A, citrinin, patulin, sterigmatocystin or aflatoxin B1 on standard agarmedia. The novel species had a high growth rate on agar media supplemented with 5% NaCl, and could bedistinguished from other Penicillium section Cinnamopurpurea species by phenotypic and molecular characteristics.

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.

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.

Species of Alternaria are serious plant pathogens, causing major losses on a wide range of crops. Leaf blight symptoms were observed on tomato leaves, and samples were collected from various regions. Isolation was done from symptomatic tomato leaves, and 15 representatives were selected from a collection of 65 isolates of Alternaria species. The virulence of Alternaria isolates was investigated on detached leaves (DL) and whole plants of tomato cv. Super strain B. A phylogenetic analysis was performed based on three partial gene regions, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the RNA polymerase second largest subunit (RPB2) and the Alternaria major allergen gene (Alt a 1). The potentiality of Alternaria isolates to produce toxins was also investigated on the basis of thin-layer chromatography (TLC). Our investigations revealed that Alternaria isolates showed different levels of virulence either on tomato plants or DL. Based on the phylogeny of three genes, Alternaria isolates encompassed two species of small-spored morphospecies: A. alternata (14 isolates) and A. arborescens (single isolate). The produced toxins varied among Alternaria isolates with tenuazonic acid (TeA) being the most abundant mycotoxin produced by most isolates. This study highlighted on other Alternaria species in Egypt that might represent a serious concern for tomato producers as causal agents of leaf blight over other species, i.e. A. solani.

Wheat, the main source of carbohydrates worldwide, can be attacked by a wide number of phytopathogenic fungi, included Alternaria species. Alternaria species commonly occur on wheat worldwide and produce several mycotoxins such as tenuazonic acid (TA), alternariol (AOH), alternariol-monomethyl ether (AME), and altenuene (ALT), provided of haemato-toxic, genotoxic, and mutagenic activities. The contamination by Alternaria species of wheat kernels, collected in Tuscany, Italy, from 2013 to 2016, was evaluated. Alternaria contamination was detected in 93 out of 100 field samples, with values ranging between 1 and 73% (mean of 18%). Selected strains were genetically characterized by multi-locus gene sequencing approach through combined sequences of allergen alt1a, glyceraldeyde-3-phosphate dehydrogenase, and translation elongation factor 1? genes. Two well defined groups were generated; namely sections Alternaria and Infectoriae. Representative strains were analyzed for mycotoxin production. A different mycotoxin profile between the sections was shown. Of the 54 strains analyzed for mycotoxins, all strains included in Section Alternaria produced AOH and AME, 40 strains (99%) produced TA, and 26 strains (63%) produced ALT. On the other hand, only a very low capability to produce both AOH and AME was recorded among the Section Infectoriae strains. These data show that a potential mycotoxin risk related to the consumption of Alternaria contaminated wheat is high.

MotivationBiodiversity research concerns with data coming from many different domains (e.g., Biology, Geography, Evolutionary Studies, Genomics, Taxonomy, Environmental Sciences, etc.) which need to be integrated for leading to valuable Biodiversity knowledge. Collecting and integrating data from so many heterogeneous resources is not a trivial task. Data are extremely scattered, heterogeneous in format and purpose, and protected in repositories of several research institutes. Driven by the widely diffused trend of the web of sharing information through aggregation of people with the same interests (social networks), and by the new type of database architecture defined as dynamic distributed federated database, we are proposing a new paradigm of data integration in the Biodiversity domain. Here we present a new approach for the development of a Knowledge Base aiming to the collection, integration and analysis of biodiversity data implemented as a product of the MBLab project.MethodsThe implementation of the Biodiversity Knowledge Base is based on the integration of several components: a robust Database Management System (IBM DB2) managing the large volume of information from public databases like GenBank, a set of GaianDB nodes [1] to manage remote private collections of biodiversity data; the IBM Federator Server to implement the general conceptual schema integrating all biodiversity databases available across remote nodes of MBLab project partners.ResultsGaianDB is a Dynamic Distributed Federated Database of sources whose growth is regulated by biologically inspired principles and graph theoretic methods. By means of the GaianDB network architecture data remains on the remote research group servers, and each database owner is responsible for its integrity, availability and sharing. Each vertex of this network is a suitable entry point receiving the user query and responding with an output aggregating different pieces of information retrieved from the different data sources spanned all over the network. To integrate GenBank molecular data in the MBLabDB we built an efficient and reliable ETL (Extraction, Transformation and Load) module, implemented with CLIPS Rule Based Programming Language. The ETL extracts information from the feature- based GenBank entries and fits them in the MBLabDB schema. Molecular data collections are structured following a Chado-like model [2], using Sequence Ontology entities and relations. This allows to retrieve data using the biological concepts expressed by the Sequence Ontology [3]. The main result of this work is the development of a standard conceptual schema and a knowledge base architecture tailored to biodiversity data collection, integration and analysis. The database is modeled on six main sections: Taxonomic, Individual, Collection, Supply chain, Experimental molecular data. Currently two biodiversity data collections have been integrated by using GaianDB: the ITEM Collection [4] located at the I

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

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

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

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