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

Mycotoxins are secondary metabolites produced by fungi that can cause adverse health effects. Due to climate change, temperatures are expected to rise and changes in rainfall patterns are foreseen. These developments may increase fungal occurrence and mycotoxin concentrations in maize. It is therefore useful to monitor mycotoxin levels in maize and record the accompanying agronomic factors and weather parameters. This paper describes a field survey in the Netherlands in which information on soil, cultivar, green manure, tillage as well as sowing, emergence, flowering and harvest dates of silage maize were collected from 148 growers. A small number of these growers (42 in total) were visited to collect maize samples revealing that 50% of the samples were contaminated with Fusarium species and mycotoxins were detected in 25% of the samples. The Fusarium species that was most commonly found was F. crookwellense followed by F. graminearum, F. culmorum, F. sporotrichiodes and F. equiseti. In total 31 mycotoxins were analysed. The predominant mycotoxins present were (sum of 3 and 15)-acetyl-DON and nivalenol; other mycotoxins found were alternariol, beauvericin, deoxynivalenol, diacetoxyscirpenol, moniliformin and zearalenone. Nivalenol was present in concentrations up to 1670 µg kg(-1) and acetylated DON was usually present at higher concentrations than DON. Statistical analysis of the current data showed no correlation between mycotoxins present and agronomic factors recorded. Field studies as described in this paper are useful and need to be continued in the future in order to observe trends in mycotoxin occurrence.

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.

Climate change has been reported as a driver for emerging food and feed safety issues worldwide and its expected impact on the presence of mycotoxins in food and feed is of great concern. Aflatoxins have the highest acute and chronic toxicity of all mycotoxins; hence, the maximal concentration in agricultural food and feed products and their commodities is regulated worldwide. The possible change in patterns of aflatoxin occurrence in crops due to climate change is a matter of concern that may require anticipatory actions. The aim of this study was to predict aflatoxin contamination in maize and wheat crops, within the next 100 years, under a +2 degrees C and +5 degrees C climate change scenario, applying a modelling approach. Europe was virtually covered by a net, 50 x 50 km grids, identifying 2254 meshes with a central point each. Climate data were generated for each point, linked to predictive models and predictions were run consequently. Aflatoxin B-1 is predicted to become a food safety issue in maize in Europe, especially in the +2 degrees C scenario, the most probable scenario of climate change expected for the next years. These results represent a supporting tool to reinforce aflatoxin management and to prevent human and animal exposure.

This study was designed to investigate the degree of aggressiveness of Fusarium graminearum sensu stricto isolates and its relationship with trichothecene production. In order to characterize Fusarium strains aggressiveness, disease severity was visually assessed as the percentage of spikelets bleached per ear. The severity ranged from a minimum of 27.19% seven days after inoculation to a maximum of 84.73% at the end of the experiment. At maturity the ears were harvested and threshed for grain weight determination. All treatments showed significant differences in kernel weight with respect to the control plants, with a yield reduction of 35–85% in comparison with the yield of the control. Grains infected by F. graminearum may contain significant levels of mycotoxins like trichothecenes. No correlation was found between aggressiveness and the toxins in the kernels at maturity, taking into consideration not only the quantity but also the type of toxin (nivalenol, deoxynivalenol or their acetylderivatives). Kernel weight reduction was a better estimator of the presence of deoxynivalenol in the kernels than the area under the disease progress curve (AUDPC) calculated with severity ratings. The amplified fragment length polymorphism (AFLP) technique was used to establish genetic relationships between 18 Argentinean isolates and eight reference strains of the Fusarium graminearum complex. All the isolates studied grouped with the two F. graminearum s. str. reference isolates, with a similarity coefficient greater than 75%. The other reference strains of the F. graminearum complex were clearly separated, with similarities ranging between 55 and 73%. The AFLP groups had no relationship with toxin accumulation on kernels or with the geographical origin of the isolates. Great heterogeneity was found in the AUDPC, yield reduction and toxin accumulation values across the regions.

Pink ear rot of maize caused by Fusarium verticillioides, Fusariumproliferatum and Fusariumgraminearum can lead to severe yield losses and contamination of grain with a range of mycotoxins. Maize stalks colonized by Fusarium spp. are the main primary inoculum source for Fusarium incited epidemics in maize or other susceptible crops grown in rotation.The colonization of individual maize stalks originating from fields in The Netherlands, Italy and Nigeria by Fusarium spp. was quantified using specific TaqMan-PCR assays. Highest values were found for F. graminearum and Fusariumavenaceum in Dutch samples, for F. graminearum and FUM producing species (F. verticillioides and F. proliferatum) in Italian samples and FUM producing Fusarium spp. in Nigerian samples. The increase in Fusarium spp. colonization under field conditions during a period of 3-6 months after harvest of the maize crops varied considerably between individual stalks. The fungal and bacterial diversity was analyzed for sub-sets of stalks with high versus low increase of Fusarium colonization. Bacterial taxonomic groups such as Bacillus, Curtobacterium, Erwinia, Flavobacterium, Novosphingobium, Pantoea, Sphingomonas, Rahnella and Staphylococcus and fungal taxonomic groups such as Acremomium sp., Cryptococcus flavescens, Cryptococcus zeae, Leptosphaeria sp. and Microdochium bolleyi were more abundant in the stalks with lower increase in pathogen colonization. Such fungal and bacterial groups associated with 'suppressive stalks' may be antagonistic to Fusarium spp. and a source of candidate strains for the selection of new biological control agents.

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.

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

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.

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.

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.

Fusarium head blight (FHB) of wheat, caused by several Fusarium species, is a damaging disease, resulting not only in yield reduction but also accumulation of mycotoxins in grain. Epidemiology and management of FHB has been extensively studied worldwide. Data on FHB development and accumulation of mycotoxins were obtained in four European countries during 2001-2004 to study the effect of FHB development and environmental conditions on accumulation of deoxynivalenol (DON). The occurrence of DON was highly correlated with presence of one or more toxigenic Fusarium species. Hourly weather data recorded at each sampling site were summarised over several periods of different lengths (5-30 days) during the anthesis and pre-harvest period. All-subsets regression was used to determine the extent to which the probability of DON occurrence is related to weather variables and also the consistency of such a toxin-weather relationship. Combined with a re-sampling technique, all-subsets regression analysis showed the difficulties in identifying a single 'best' model of relating the probability of toxin a parts per thousand yen90 mu g kg(-1) to weather predictors. A wide range of inter-related weather predictors based on time windows around anthesis and pre-harvest were selected in different models. There were many alternative models based on weather predictors only with similar predictive power because of high correlation among weather predictors. The performance of these alternative models was generally poor, particularly in terms of the high proportion of false positive predictions (specificity was only around 0.60-0.65). Inclusion of the number of toxigenic Fusarium species at harvest into models did not improve the model sensitivity (ca. 0.75-0.80) but appreciably improved the specificity (ca. 0.70-0.75). On balance, weather summarised over a 15-day window frame led to models with better predictions than other three window frames (5, 10 and 30 days).

Fusarium culmorum, a pathogenic fungal species associated with Fusarium Head Blight (FHB) of wheat, can produce the mycotoxin deoxynivalenol (DON) that is frequently found as contaminant in cereals. Wheat haulms, as decomposing plant material, are an important inoculum source of F. culmorum for subsequent crops. In the present work we exploited the mycobiota of haulms buried in natural soil as a source of potential antagonists of F. culmorum and evaluated the role played by DON in determining the composition of such mycobiota. DON was added to wheat haulm pieces that were incubated in three natural soils, namely sandy, clayey and mixed, all with a previous history of wheat cultivation. Composition of mycobiota associated with DON-treated cultural debris was evaluated and compared with mycobiota from control haulms, in order to evaluate the effect of soil type and of the presence of the mycotoxin. Our results showed that DON affected neither the number nor species profile of fungal isolates. Among fungi associated with cultural debris, thirty-nine Pythium spp. isolates were collected and five of them, both from treated and not treated haulms, were tested for their antagonistic ability against F. culmorum. The two strains showing the highest antagonistic activity were further tested against F. oxysporum, a species considered a major competitor of F. culmorum for wheat residues, and frequently recovered from haulms in our experiments. The two Pythium spp. isolates showed a positive antagonistic and mycoparasitic activity against F. culmorum, without affecting the growth of F. oxysporum. Mycobiota of haulms buried in soil represents an interesting source of potential antagonists and competitors for future exploitation in a multitrophic approach for biocontrol of FHB.

Fusarium poae is one of the Fusarium species commonly detected in wheat kernels affected by Fusarium Head Blight. Fusarium poae produces a wide range of mycotoxins including nivalenol (NIV). The effect of temperature on colony growth and NIV production was investigated in vitro at 5-40 degrees C with 5 degrees C intervals. When the data were fit to a Beta equation (R-2 >= 0.97), the optimal temperature was estimated to be 24.7 degrees C for colony growth and 27.5 degrees C for NIV production. The effects of temperature on infection incidence, fungal biomass, and NIV contamination were investigated by inoculating potted durum wheat plants at full anthesis; inoculated heads were kept at 10-40 degrees C with 5 degrees C intervals for 3 days and then at ambient temperature until ripening. Temperature significantly affected the incidence of floret infection and fungal biomass (as indicated by DNA amount) in the affected heads but did not affect NIV content in the head tissue. Inoculation of potted plants with F. poae did not reduce yield. (c) 2018 Elsevier Ltd. All rights reserved.

The efficacy of agrochemical treatments, based on three different fungicides combined with an insecticide,was tested in southern Italy for two years on three maize hybrids to control Fusarium ear rotof maize and the accumulation in the maize kernels of the carcinogenic mycotoxins fumonisins. Insectdamage incidence and severity, disease incidence and severity, identification of Fusarium species andlevels of fumonisin contamination in kernels were determined. Field trials showed in both years thatnatural colonization of maize kernels by the fumonisin producing species Fusarium proliferatum and F.verticillioides (up to 81.5 and 26.5%, respectively) and total fumonisin contamination (up to 68.2gg-1)were highly severe. For all hybrids and in both years, the treatment with the insecticide applied alonereduced the insect damage severity consistently and the content of fumonisins in the kernel only in halfof the cases, whereas fungicide treatments applied in combination with the insecticide showed a furthersignificant reduction of fumonisin contamination in the three hybrids and in both years.

Descrizione del Progetto "EZIOCONTROL": obiettivi, metodologie di ricerca, risultati

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.

This review summarizes the information on biochemical and biological activity of the main Fusarium mycotoxins, focusing on toxicological aspects in terms of species-specific effects. Both in vitro and in vivo studies have centered on the peculiarity of the responses to mycotoxins, demonstrating that toxicokinetics, bioavailability and the mechanisms of action of these substances vary depending on the species involved, but additional studies are needed to better understand the specific responses. The aim of this review is to summarize the toxicological responses of the main species affected by Fusarium mycotoxins.

Fusarium head blight is a disease caused by a complex of Fusarium species. F. poae is omnipresent throughout Europe in spite of its low virulence. In this study, we assessed a geographically diverse collection of F. poae isolates for its genetic diversity using AFLP (Amplified Fragment Length Polymorphism). Furthermore, studying the mating type locus and chromosomal insertions, we identified hallmarks of both sexual recombination and clonal spread of successful genotypes in the population. Despite the large genetic variation found, all F. poae isolates possess the nivalenol chemotype based on Tri7 sequence analysis. Nevertheless, Tri gene clusters showed two layers of genetic variability. Firstly, the Tri1 locus was highly variable with mostly synonymous mutations and mutations in introns pointing to a strong purifying selection pressure. Secondly, in a subset of isolates, the main trichothecene gene cluster was invaded by a transposable element between Tri5 and Tri6. To investigate the impact of these variations on the phenotypic chemotype, mycotoxin production was assessed on artificial medium. Complex blends of type A and type B trichothecenes were produced but neither genetic variability in the Tri genes nor variability in the genome or geography accounted for the divergence in trichothecene production. In view of its complex chemotype, it will be of utmost interest to uncover the role of trichothecenes in virulence, spread and survival of F. poae.

The Fusarium graminearum species complex (FGSC) is a group of mycotoxigenic fungi that are the primary cause of Fusarium head blight (FHB) of wheat worldwide. The distribution, frequency of occurrence, and genetic diversity of FGSC species in cereal crops in South America is not well understood compared to some regions of Asia, Europe and North America. Therefore, we examined the frequency and genetic diversity of a collection of 183 FGSC isolates recovered from wheat grown during multiple growing seasons and across a large area of eastern Argentina, a major wheat producing region in South America. Sequence analysis of the translation elongation factor 1-alpha and beta-tubulin genes as well as Amplified Fragment Length Polymorphism (AFLP) analyses indicated that all isolates were the FGSC species F. graminearum sensu stricto. AFLP analysis resolved at least 11 subgroups, and all the isolates represented different AFLP haplotypes. AFLP profile and geographic origin were not correlated. Previously obtained trichothecene production profiles of the isolates revealed that the 15-acetyldeoxynivalenol chemotype was slightly more frequent than the 3-acetyldeoxynivalenol chemotype among the isolates. These data extend the current understanding of FGSC diversity and provide further evidence that F. graminearum sensu stricto is the predominant cause of FHB in the temperate main wheat-growing area of Argentina. Moreover, two isolates of F. crookwellense and four of F. pseudograminearum were also recovered from wheat samples and sequenced. The results also suggest that, although F. graminearum sensu stricto was the only FGSC species recovered in this study, the high level of genetic diversity within this species should be considered in plant breeding efforts and development of other disease management strategies aimed at reducing FHB.

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.

Fusarium proliferatum is together with Fusarium verticillioides the main source of fumonisins, a health riskmycotoxin, contaminating agro-products. Contrary to F. verticillioides, it colonizes a wide range of hostplants besides maize, such as wheat or barley among others, in particular in certain regions (SouthernEurope). The phylogenetic study performed in this work using a wide sample of isolates from diverse hostsand origins revealed a high variability, while no host preferences could be sustained. A real time RT-PCRassay was also developed specific for F. proliferatum on the basis on fumonisin biosynthetic gene, FUM1,which allowed discrimination from F. verticillioides. FUM1 gene expression showed a high and significantcorrelation (0.77) with fumonisin production, representing a valuable tool for specific and sensitive diagnosisof metabolically active fumonisin-producing F. proliferatum isolates and for evaluating the influenceon environmental conditions on FUM1 gene regulation. The ability to produce fumonisins was also widelydistributed indicating that F. proliferatum can represent a risk for health similarly to F. verticillioides.Moreover, the wide range of plants susceptible to colonization by F. proliferatum suggests that the impact offumonisin risk in a number of commodities might need a revision.

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.

Fusarium graminearum is among the main causal agents of Fusarium head blight (FHB), or scab, of wheat and other cereals, caused by a complex of Fusarium species, worldwide. Besides causing economic losses in terms of crop yield and quality, F. graminearum poses a severe threat to animal and human health. Here, we present the first draft whole-genome sequence of the mycotoxigenic Fusarium graminearum strain ITEM 124, also providing useful information for comparative genomics studies.

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

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.

Microbial volatile organic compounds (MVOCs) were collected in water-damaged buildings to evaluate their use as possible indicators of indoor fungal growth. Fungal species isolated from contaminated buildings were screened for MVOC production on malt extract agar by means of headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) analysis. Some sesquiterpenes, specifically derived from fungal growth, were detected in the sampled environments and the corresponding fungal producers were identified. Statistical analysis of the detected MVOC profiles allowed the identification of species-specific MVOCs or MVOC patterns for Aspergillus versicolor group, Aspergillus ustus, and Eurotium amstelodami. In addition, Chaetomium spp. and Epicoccum spp. were clearly differentiated by their volatile production from a group of 76 fungal strains belonging to different genera. These results are useful in the chemotaxonomic discrimination of fungal species, in aid to the classical morphological and molecular identification techniques.

A Penicillium polonicum, an Aspergillus ustus and a Periconia britannica strain were isolated from water-damaged environments and the production of microbial volatile organic compounds (MVOCs) was investigated by means of headspace solid-phase microextraction followed by GC-MS analysis. The most important MVOCs produced were 2-methylisoborneol, geosmin and daucane-type sesquiterpenes for P. polonicum, 1-octen-3-ol, 3-octanone, germacrene D, ?-cadinene and other sesquiterpenes for A. ustus and the volatile mycotoxin precursor aristolochene together with valencene, ?-selinene and ?-selinene for P. britannica. Different growth conditions (substrate, temperature, relative humidity) were selected, resembling indoor parameters, to investigate their influence on fungal metabolism in relation with the sick building syndrome and the results were compared with two other fungal strains previously analyzed under the same conditions. In general, the range of MVOCs and the emitted quantities were larger on malt extract agar than on wallpaper and plasterboard, but, overall, the main MVOC profile was conserved also on the two building materials tested. The influence of temperature and relative humidity on growth and metabolism is different for different fungal species, and two main patterns of behavior could be distinguished. Results show that, even at suboptimal conditions for growth, production of fungal volatiles can be significant.

The management of good agricultural practices during pre-harvest is a key issue for minimising the risk of Fusarium mycotoxin accumulation in the crops. Such practices involve crop rotation, tillage, appropriate fertilisation and fungicide application, use of biological control agents, variety selection, timely planting and harvests as well as control of insects, which frequently facilitate Fusarium species infections. On the other hand, the reduction of Fusarium mycotoxins along the malting and brewing chain is also highly dependent on a correct post-harvest and malting management that must aim firstly at the separation of the infected crop products from the healthy material. Therefore, the use of different tools such as manual sorting or optical sensors is also a crucial aspect for reducing the level of mycotoxin contamination. Moreover, it is extremely important to prevent post-harvest contamination and develop practical and effective post-harvest procedures for mycotoxin reduction in the food supply chains and to provide alternative and safe use options for contaminated batches. An updated review will be given on integrated management of pre-and post-harvest practices aiming at minimising the risk of mycotoxin contamination along the malting and brewing chain and main effective solutions, including the development of a MycoKey app, proposed and to be achieved within the EU project MycoKey (http://www.mycokey.eu/) .

Trichoderma gamsii T6085 was used in combination with a Fusarium oxysporum isolate (7121) in order to evaluate, in a multitrophic approach, their competitive ability against F. graminearum, one of the main causal agents of Fusarium head blight (FHB) on wheat. The two antagonists and the pathogen were coinoculated on two different natural substrates, wheat and rice kernels. Both T6085 and 7121, alone and coinoculated, significantly reduced the substrate colonization and mycotoxin production by the pathogen. The two antagonists did not affect each other. Using a metabolic approach (Biolog), we investigated whether exploitation competition could explain this antagonistic activity. The aim was to define whether the three fungi coexist or if one isolate nutritionally dominates another. Results obtained from Biolog suggest that no exploitative competition occurs between the antagonists and the pathogen during the colonization of the natural substrates. Interference competition was then preliminarily evaluated to justify the reduction in the pathogen's growth and to better explain mechanisms. A significant reduction of F. graminearum growth was observed when the pathogen grew in the cultural filtrates of T. gamsii T6085, both alone and cocultured with F. oxysporum 7121, thus suggesting the involvement of secondary metabolites. As far as we know, this is the first time that an ecological study has been performed to explain how and which kind of competition could be involved in a multitrophic biocontrol of FHB.

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/).

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.

Fusarium diseases of Liliaceae crops cause significant losses worldwide. Yet some Fusarium species are found in planta without causing disease or even in a symbiotic relationship with its host. In this study we identified and characterized the Fusarium species isolated from soil, and from healthy and diseased bulbs of Lilium longiflorum grown in New Jersey and Oregon in the United States. The predominant Fusarium species from the Oregon location were F. solani (74%) and F. oxysporum (20%), whereas F. concentricum (43%) and F. proliferatum (26%), both belonging to the Gibberella fujikuroi species complex (GFSC), were the most commonly isolated species from New Jersey. To our knowledge, this is the first report of F. concentricum associated with Liliaceae. All of the isolates were characterized with sequences of the internal transcribed spacer and translation elongation factor 1-alpha genes. The 24 GFSC isolates were further characterized with mating type, mating population, and mycotoxin analysis. Results showed that all GFSC isolates were MAT-2, suggesting that the populations may be asexually reproducing in the region examined. The majority of the GFSC isolates produced beauvericin. Enniatin A, B, B(1) and fusaproliferin were produced by a few isolates. Enniatin A(1) and fumonisins were not detected in any of the isolates. Although F. oxysporum and F. solani are well-known bulb pathogens, many isolates of F. oxysporum and F. solani, and all of the F. concentricum and F. proliferatum were isolated from asymptomatic bulbs, suggesting their endophytic association with lilies.

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.

As determined by the Intergovernmental Panel on Climate Change, warming of the climate system is unequivocal and has been associated with rising sea levels, diminished amounts of ice and snow and increasing oceanic and atmospheric temperatures. Such climate changes have a significant impact on stages and rates of toxigenic fungi development and can modify host-resistance and host-pathogen interactions, influencing deeply also the conditions for mycotoxin production that vary for each individual pathogen. Moreover, the new combinations mycotoxins/host plants/geographical areas are arising to the attention of the scientific community and require new diagnostic tools and deeper knowledge of both biology and genetics of toxigenic fungi. In this review, it is underlined that an extension of the aflatoxin contamination risk in maize in South and Central-Europe is highly likely in the next 30 years, due to favorable climatic conditions to the growth of Aspergillus flavus. Moreover, the mycotoxigenic Fusarium species profile on wheat in Europe is in continuous change in Northern, Central and Southern-Europe with, in particular, a worrisome growing contamination of F. graminearum in the Central and Northern Europe.

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.

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

This study aimed to monitor the main toxigenic fungi in neighbouring organic and conventional maize and wheat fields in Italy in 2010 and 2011. The Fusarium species mainly isolated were: Fusarium poae, sometimes predominant on Fusarium graminearum in wheat, and Fusarium verticillioides competing with Fusarium proliferatum and Fusarium subglutinans in maize. The incidence of Fusarium spp. was similar for both conventional (6%) and organic (4%) wheat, but it was influenced by weather conditions. 2010 was the most favourable for Fusarium species, with 10 times the incidence of 2011. Fusarium infection was significantly different between farming systems in maize (20% vs 35% in conventional and organic, respectively), while in 2010 the incidence was significantly higher than in 2011 (43% vs 25%). Aspergillus and Penicillium incidence was not linked to the farming system but to weather conditions, with moderately higher incidence in 2010. (C) 2015 Elsevier Ltd. All rights reserved.

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.

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.

Fusarium equiseti and Fusarium acuminatum are toxigenic species that contaminate cereal crops from diverse climatic regions. They are common in Spanish cereals. The information available on their phylogenetics and toxigenic profiles is, however, insufficient to assist risk evaluation. In this work, phylogenetic analyses were performed using partial sequences of the translation elongation factor gene (EF-1?) of F. equiseti and F. acuminatum strains isolated from barley and wheat from Spain and other countries. The Northern and Southern European F. equiseti strains largely separated into two phylogenetically distinct clusters. This suggests the existence of two distinct populations within this species, explaining its presence in these regions of markedly different climate. Production of type A and B trichothecenes by the Spanish strains, examined in wheat cultures using a multitoxin analytical method, indicated that F. equiseti could produce deoxynivalenol and nivalenol and other trichothecenes, at concentrations that might represent a significant risk of toxin contamination for Southern European cereals. F. acuminatum showed low intraspecific genetic variability and 58% of the strains could produce deoxynivalenol at low level. Neither species was found to produce T-2 or HT-2 toxins. The present results provide important phylogenetic and toxigenic information essential for the accurate prediction of toxigenic risk.

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.

Surveys for crown rot (FCR) and head blight (FHB) of Algerian wheat conducted during 2014 and 2015 revealed that Fusarium culmorum strains producing 3-acetyl-deoxynivalenol (3ADON) or nivalenol (NIV) were the causal agents of these important diseases. Morphological identification of the isolates (n FCR = 110, n FHB = 30) was confirmed by sequencing a portion of TEF1. To assess mating type idiomorph, trichothecene chemotype potential and global population structure, the Algerian strains were compared with preliminary sample of F. culmorum from Italy (rt = 27), Australia (n = 30) and the United States (n = 28). A PCR assay for MAT idiomorph revealed that MAT1-1 and MAT1-2 strains were segregating in nearly equal proportions, except within Algeria where twothirds of the strains were MAT1-2. An allele-specific PCR assay indicated that the 3ADON trichothecene genotype was predominant globally (83.8% 3ADON) and in each of the four countries sampled. In vitro toxin analyses confirmed trichothecene genotype PCR data and demonstrated that most of the strains tested (77%) produced culmorin. Global population genetic structure of 191 strains was assessed using nine microsatellite markers (SSRs). AMOVA of the clone corrected data indicated that 89% of the variation was within populations. Bayesian analysis of the SSR data identified two globally distributed, sympatric populations within which both trichothecene chemotypes and mating types were represented.

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

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

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

Nowadays, complaints about poor indoor air quality have become common. The variety of indoor-air health problems include chronic fatigue, allergy, skin and eye irritation and can be caused by several factors including fungi and their metabolites present in a building. The objective of this study was to establish a simple method for untargeted analysis of secondary fungal metabolites in indoor environments. As a detection technique, it was opted for time-of-flight mass spectrometry, as it provided high sensitivity and mass accuracy in full scan acquisition mode. The method was firstly applied to fungal cultures, namely Penicillium brevicompactum and Chaetomium murorum, which were isolated from mouldy houses and grown on building materials in laboratory conditions. Following the proposed strategy based on accurate mass measurement and post-acquisition data processing using statistical tools, a number of metabolites were identified. Subsequently, samples from mouldy inhabited buildings were screened using the developed method.

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.