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

Laccases (LCs) are multicopper oxidases that find application as versatile biocatalystsfor the green bioremediation of environmental pollutants and xenobiotics. In this study weelucidate the degrading activity of Lac2 pure enzyme form Pleurotus pulmonarius towards aflatoxinB1 (AFB1) and M1 (AFM1). LC enzyme was purified using three chromatographic steps andidentified as Lac2 through zymogram and LC-MS/MS. The degradation assays were performedin vitro at 25 °C for 72 h in buffer solution. AFB1 degradation by Lac2 direct oxidationwas 23%. Toxin degradation was also investigated in the presence of three redox mediators,(2,20-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) (ABTS) and two naturally-occurring phenols,acetosyringone (AS) and syringaldehyde (SA). The direct effect of the enzyme and the mediatedaction of Lac2 with redox mediators univocally proved the correlation between Lac2 activity andaflatoxins degradation. The degradation of AFB1 was enhanced by the addition of all mediators at10 mM, with AS being the most effective (90% of degradation). AFM1 was completely degraded byLac2 with all mediators at 10 mM. The novelty of this study relies on the identification of a pureenzyme as capable of degrading AFB1 and, for the first time, AFM1, and on the evidence that themechanism of an effective degradation occurs via the mediation of natural phenolic compounds.These results opened new perspective for Lac2 application in the food and feed supply chains as abiotransforming agent of AFB1 and AFM1

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

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.

Ochratoxin A (OTA) is a mycotoxin with a main nephrotoxic activity contaminatingseveral foodstuffs. In the present report, five soil samples collected from OTA-contaminatedvineyards were screened to isolate microorganisms able to biodegrade OTA. When cultivated inOTA-supplemented medium, OTA was converted in OTalpha by 225 bacterial isolates. To revealclonal relationships between isolates, molecular typing by using an automated rep-PCR systemwas carried out, thus showing the presence of 27 different strains (rep-PCR profiles). The16S-rRNA gene sequence analysis of an isolate representative of each rep-PCR profiles indicatedthat they belonged to five bacterial genera, namely Pseudomonas, Leclercia, Pantoea, Enterobacter, andAcinetobacter. However, further evaluation of OTA-degrading activity by the 27 strains revealedthat only Acinetobacter calcoaceticus strain 396.1 and Acinetobacter sp. strain neg1, consistentlyconserved the above property; their further characterization showed that they were able to convert82% and 91% OTA into OTalpha in six days at 24 °C, respectively. The presence of OTalpha, asthe unique OTA-degradation product was confirmed by LC-HRMS. This is the first report onOTA biodegradation by bacterial strains isolated from agricultural soils and carried out underaerobic conditions and moderate temperatures. These microorganisms might be used to detoxifyOTA-contaminated feed and could be a new source of gene(s) for the development of a novelenzymatic detoxification system.

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

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.

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

Ochratoxin A (OTA) is a mycotoxin denoted by a nephrotoxic activity contaminating several foodstuffs. Nowadays, the biological systems for OTA degradation to the less toxic OT? aroused great interest by the scientific community. In the present study, bacteria able to biodegrade OTA were isolated from soil samples collected in OTA-contaminated vineyards. Soil samples were collected from five vineyards of Negroamaro and Primitivo grape cultivars in Salento (Southern Italy). They were cultured in appropriate media added with OTA, mycotoxin degradation was determined by HPLC/FLC analysis and bacterial colonies were isolated by plating. Clonal relationships between isolates was assessed by using an automated rep-PCR system and then each strain was identified by 16S rRNA gene sequencing. A total of 225 bacterial isolates were able to convert OTA in OT?. The molecular analysis of the above isolates showed the presence of 27 different strains (rep-PCR profiles). The sequence analysis of the 16S-rRNA gene indicated that they belonged to five bacterial genera: Pseudomonas, Leclercia, Pantoea, Enterobacter and Acinetobacter. Additional assessment of OTA-degrading capacity of the 27 strains indicated that only the Acinetobacter calcoaceticus strain 396.1 and the A. sp. strain neg1 conserved the above property: both strains were further studied thus showing that they were able to convert 82% and 91% OTA into OT? in 6 days at 24°C, respectively. The occurrence of OT?, as the sole OTA-degradation product was established by LC-MS/MS.This is the first description on OTA biodegradation under aerobic conditions and moderate temperature by bacterial strains from agricultural soils. These microorganisms might be used to detoxify OTA contaminated feed and could be a resource for the development of a new enzymatic detoxification system.

The effect of processing on mycotoxin content in milling fractions has been investigated in 10 samples of durum wheat contaminated with T-2 and HT-2 toxins at levels ranging from 97 to 5,954 ?g/kg (sum of T-2 and HT-2 toxins). Either naturally contaminated samples or samples artificially inoculated with Fusarium sporotrichioides under field conditions were used. A method based on liquid chromatography-tandem mass spectrometry coupled with immunoaffinity column cleanup was validated in-house for the simultaneous analysis of both toxins in a variety of matrices, including uncleaned wheat, cleaned wheat, screenings, bran, red dog, fine middlings, and semolina. Mean recoveries from samples spiked with T-2 and HT-2 toxins at levels of 100 ?g/kg ranged from 85 to 107%, with relative standard deviations (RSDs) lower than 14%. The milling process led to an increase of T-2 and HT-2 toxin contents up to 13-and 5-fold in screenings and bran, respectively, compared with occurrence in the uncleaned wheat; however, an overall reduction of T-2 and HT-2 toxins by 54% (RSD, 20%) and 89% (RSD, 3%) was observed in cleaned wheat and in semolina, respectively. Copyright © International Association for Food Protection.

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.

Ochratoxin A (OTA) is a nephrotoxic and potentially carcinogenic mycotoxin produced by several species of Aspergillus and Penicillium. It is one of the major mycotoxins contaminating grain, grapes and a variety of food products, and the development of methods for reducing pre-and post-harvest contamination has drawn considerable attention. In the current study, we isolated and sequenced the genome of a novel free-living Acinetobacter strain able to degrade OTA. Biochemical studies suggest that the degradation reaction proceeds via peptide bond hydrolysis.

Grains and grain-based products provide the largest contribution to the sum of HT-2 and T-2 toxin exposure for humans and their monitoring is of high priority. The European Commission has recently published a recommendation on the presence of T-2 and HT-2 toxins in cereals and cereal products. An indicative level of 50 ?g/kg has been fixed for the sum of T-2 and HT-2 toxins in durum wheat (Triticum turgidum ssp. durum Desf.) and other cereals for direct human consumption. Wheat debranning, a process by which outer kernel layers are removed through both friction and abrasive scouring actions, positively affects several quality features of semolina such as the improvement of milling yield and the reduction of heat damage. Moreover, the same process provides a powerful tool to improve hygienic-sanitary quality of derived products. Aim of the present study was to evaluate the effectiveness of debranning process to reduce the levels of T-2 and HT-2 toxins in treated kernels and milling fractions. A durum wheat sample contaminated with T-2 and HT-2 toxins at level of 122 ?g/kg (sum of T-2 and HT-2 toxins) was subjected to two consecutive debranning steps at laboratory scale, each one of the duration of 30s. Debranned and not-debranned kernels were processed by a pilot milling plant. All samples, including debranned kernels, debranning wastes and milling fractions (semolina, coarse bran, fine bran and fine middlings) were analysed for the sum of T-2 and HT-2 toxins by enzyme-linked immunoassay (Veratox® for T-2/HT-2, Neogen) and by LC-MS/MS after immunoaffinity column clean up. Results showed that the debranning process significantly reduced levels of T-2 and HT-2 toxins in the kernels with a reduction rate of 72% after the first 30s step. No significant toxin reduction was observed after the second 30s debranning step. High mycotoxin levels were found in the debranning wastes. The semolina fractions, after 0.4 mm sieving, obtained from both debranned and unprocessed kernels showed not significant differences in toxin content, suggesting an accumulation of these toxins in the most peripheral layers. Interestingly, coarse bran obtained from debranned kernels at first step (30s) resulted to be less contaminated (201 µg/kg) than that obtained from unprocessed kernels (433 µg/kg). Preliminary results obtained in the present study indicate that the debranning process is useful to reduce T-2 and HT-2 toxin contamination present on the surface of the wheat kernel and could play an important role to obtain whole-wheat products or less refined products with high nutritional value and hygienic-sanitary quality.

At the three-leaf stage, sterile seedlings of resistant (CO433) and susceptible (CO389) maize lines was spiked with fumonisin B1 (FB1) (dissolved in PBS, pH =7.4, at the final concentration of 1mg/mL) in the part of the stem between the collar and the insertion of the first leaf. At various times, the FB1 content was determined by the use of HPLC/FLD previously derivatized with an o-phthaldeyde (OPA). The stem was ground with liquid nitrogen and extracted with a solution methanol/water (70:30, v/v). Recovery was 106% (RSD < 8%). The concentration of FB1 after 3 hours (88%, RSD 12%) and 48 hours (92%, RSD 7%) after spiking showed that no translocation in seedling maize occurred. To evaluate if the defence systems were alerted, the leaves were used to monitor the ascorbate-glutathione cycle, phenolics and enzymes protective from oxidative stress (catalase, superoxide dismutase and cytosolic peroxidases) at 3 and 48 hours post-spiking. The study was also extended to the analysis of total antioxidants, hydrogen peroxide and malondialdehyde contents to evaluate the oxidation level after FB1 treatment. Defense response promptly appeared activated in leaves of resistant line; particularly, after 3h, ascorbate, ascorbate peroxidase, SOD were augmented, underlining a higher fitness in the counteracting the phytotoxic action of FB1. In contrast, in the susceptible line, catalase, phenolics and ascorbate increased at longer time, conferring a lower readiness to the FB1 treatment. Same trend in total antioxidants, cytosolic peroxidases and other components analyzed was observed in both CO433 and CO389 after spiking. FB1, although did not cause seedlings suffering, induced metabolic perturbations. These data are useful for further investigation on molecular mechanisms that are to the basis of the FB1 and others mycotoxins contamination in maize, in order to improve resistance to fungal pathogens.

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

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.

I cereali e i prodotti a base di cereali costituiscono la maggiore fonte di esposizione dell'uomo alle tossine T-2 e HT-2, metaboliti tossici prodotti da funghi del genere Fusarium. Lo scopo del presente lavoro è stato quello di valutare l'effetto del processo di decorticazione sul contenuto delle tossine T-2 e HT-2 nella granella e nelle frazioni di macinazione del frumento duro. Le analisichimiche hanno evidenziato un'efficace azione del processo di decorticazione nel ridurre i livelli di tossine T-2 e HT-2 nella granella e nelle principali frazioni di scarto di macinazione ad elevato valore nutritivo (crusca e farinetta), suggerendo una distribuzione delle due micotossine limitata ai tegumenti esterni delle cariossidi. I livelli di contaminazione nelle semole setacciate ottenute sia dai campioni decorticati, sia tal quali, sono risultati trascurabili.

Members of the fungal genus Fusarium can produce numerous secondary metabolites, including the nonribosomal mycotoxins beauvericin (BEA) and enniatins (ENNs). Both mycotoxins are synthesized by the multifunctional enzyme enniatin synthetase (ESYN1) that contains both peptide synthetase and S-adenosyl-l-methionine-dependent N-methyltransferase activities. Several Fusarium species can produce ENNs, BEA or both, but the mechanism(s) enabling these differential metabolic profiles is unknown. In this study, we analyzed the primary structure of ESYN1 by sequencing esyn1 transcripts from different Fusarium species. We measured ENNs and BEA production by ultra-performance liquid chromatography coupled with photodiode array and Acquity QDa mass detector (UPLC-PDA-QDa) analyses. We predicted protein structures, compared the predictions by multivariate analysis methods and found a striking correlation between BEA/ENN-producing profiles and ESYN1 three-dimensional structures. Structural differences in the ? strand's Asn789-Ala793 and His797-Asp802 portions of the amino acid adenylation domain can be used to distinguish BEA/ENN-producing Fusarium isolates from those that produce only ENN.

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

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.

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.

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

The presence of glucoside derivatives of T-2 and HT-2 toxins (type A trichothecene mycotoxins) in naturally contaminated wheat and oats is reported for the first time. The use of advanced high-resolution mass spectrometry based on Orbitrap technology allowed to obtain molecular structure details by measuring exact masses of main characteristic fragments, with mass accuracy lower than 2.8 ppm (absolute value). A monoglucoside derivative of T-2 toxin and two monoglucoside derivatives of HT-2 toxin were identified and characterized. The analysis of their fragmentation patterns provided evidence for glucosylation at C-3 position for T-2 toxin and at C-3 or C-4 position for HT-2 toxin. A screening for the presence of these new masked forms of mycotoxins was carried out on a set of naturally contaminated wheat and oats samples. On the basis of peak area ratio between glucoside derivatives and free T-2 and HT-2 toxins, the presence of glucoside derivatives was more likely in wheat than in oats samples. The present work confirms the widespread occurrence of trichothecene glucosides in cereal grains naturally contaminated with the relevant unconjugated toxins, thus suggesting the importance of developing suitable analytical methods for their detection. Besides toxicity studies, tracking down these new masked forms of trichothecenes along the food/feed chain would enable to collect information on their relevance in human/animal exposure to mycotoxin risk.

Mycotoxin contamination of staple food commodities is a relevant health and economic issue worldwide.The development of green and effective reduction strategies to counteract the contamination by multiplemycotoxins has become an urgent need. The aim of this work was to evaluate the capability of a laccase(LC) from Pleurotus eryngii and a laccase-mediator systems (LMSs) to degrade aflatoxin B1 (AFB1),fumonisin B1 (FB1), ochratoxin A (OTA), deoxynivalenol (DON), Zearalenone (ZEN) and T-2 toxin in in vitroassays. In addition, the simultaneous mycotoxin degradation capability with selected LMSs was evaluatedwith combinations of AFB1 and ZEN, and FB1 and T-2 toxin. Redox mediators were found to drasticallyincreasethe degradation efficiencies of the enzyme. AFB1, FB1, OTA, ZEN and T-2 toxin degradation by thebest performing LMS were 73%, 74%, 27%, 100% and 40%, respectively. No degradation was registered forDON. Notably, AFB1 and ZEN were simultaneously degraded by 86% and 100%, while FB1 and T-2 by 25%and 100%, respectively. LMS proved to be a promising approach to enhance degradation properties of LCenzymes and for the potential development of a multi-mycotoxin reducing method.

Powdery mildew (PM), caused by the fungus Erysiphe necator, is one of the most widespread fungal disease of grape and maycause extensive openings on the berry surface during the infection. We evaluated the effect of damage caused by PM in grape berrieson the growth of and mycotoxin production by Aspergillus and on the oxidative stress in infected berries. Berries of Vitis vinifera L.cv. Negroamaro with sound skin (SS) and those naturally infected by PM were surface sterilized and inoculated with eitherfumonisin B2 (FB2)-producing strains of Aspergillus niger or ochratoxin A (OTA)-producing strains of Aspergillus carbonariusand incubated at 20 and 30uC. The PM berries were significantly more susceptible to both Aspergillus colonization (5 to 15 timesmore susceptible) and OTA and FB2 contamination (2 to 9 times more susceptible) than were SS berries. The highest toxinconcentration was detected in inoculated PM berries both for OTA (9 ng/g) at 20uC and for FB2 (687 ng/g) at 30uC. In inoculated SSand PM berries, although malondialdehyde and hydrogen peroxide concentrations did not increase, the two black Aspergillusspecies caused a significant decrease in ascorbate content, thus inducing a pro-oxidant effect. These results indicate that grape berriesaffected by PM are more susceptible to black Aspergillus growth and to production and/or accumulation of FB2 and OTA. Thus,preventive control of E. necator on grape berries could reduce the mycotoxicological risk from black Aspergillus infection.

A series of field experiments has been conducted in North West Italy over a period of 3 years to evaluate the effect of fungicide applications on common wheat yield and safety, combined with different agronomic conditions (high: a susceptible variety to FHB planted in an untilled field; low: a medium tolerant variety to FHB planted in a ploughed field) for Fusarium head blight (FHB) infection risk. A azole mixture (prochloraz + epoxiconazole), applied at heading, was compared in each year and for each agronomic condition with a triazole with high activity against F. graminearum and F. culmorum (metconazole), a strobilurin-azole mixture with elevated action to control leaf diseases and delay leaf senescence (azoxystrobin + prochloraz) and an untreated control. The following parameters were analyzed: flag leaf greenness, grain yield, test weight, thousand kernel weight (TKW), FHB incidence and severity and deoxynivalenol (DON) contamination. The results of this research, conducted over three years with different meteorological conditions, underline the important link between fungicide efficacy and environmental and agronomic conditions that can influence fungal disease pressure. The fungicide effect on the control of FHB and the increase in flag leaf longevity and grain yield were greater with an increase in the disease pressure. On the other hand, the DON contamination was reduced by the fungicide to a greater extent in the low risk agronomic and environmental conditions compared to the high risk ones. Prochloraz + epoxiconazole showed a lower efficacy in reducing DON contamination compared to metconazole, particularly in the higher FHB pressure conditions. No significant differences were observed between the azole mixture and the strobilurin-azole mixture for flag leaf greenness, grain yield, test weight or TKW. This study provides useful information that can be used to evaluate, in naturally-infected field conditions, the effect of a fungicide application at heading on wheat yield and safety in distinct scenarios for FHB and foliar disease pressure. © M. Blandino et al., 2011 Licensee PAGEPress, Italy.

Aspergillus niger is a fungus able to produce the carcinogenic mycotoxins ochratoxin A (OTA) and fumonisins. We analysed the influence of light of various wavelengths on growth, conidiation, fumonisin B2 (FB2) and OTA biosynthesis by A. niger ITEM 7097. Light from both sides of the spectrum, from long (627 nm) to short wavelengths (470-455 nm), had a stimulating effect on growth, with the highest stimulation under blue (455 nm, 1,700 Lux) and short-wave blue light (390 nm). Conidiation was reduced by 40% under a short blue wavelength (455 nm, 200 Lux), but strongly promoted under light at an even shorter wavelength (390 nm), with an increase of about 200 fold in comparison to the dark. Production of FB2 and OTA was mutually regulated by light. FB2 production was promoted under light conditions: red and blue light in particular increased FB2 biosynthesis by 40%. Conversely, OTA production was greatly inhibited under red and blue light in comparison to dark incubation, with a mean reduction of about 40 fold, indicating a reverse regulation of both biosynthetic pathways. Incubation under a 390 nm wavelength repressed the production of both toxins to non-detectable levels.

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

Fumonisins are a group of mycotoxins, mainly found in maize and maize-based food and feed, associated with several diseases in animals. The impact of these toxins on the economy and health worldwide has driven several efforts to clarify the role of environmental factors that can influence fumonisin biosynthesis by the toxigenic species. We analyzed the influence of light of varying wavelength on growth and fumonisin biosynthesis by the fungus Fusarium proliferatum ITEM 1719. Light in general had a positive influence on growth, with a mean increase of the grow rate of about 40% under light exposure in comparison to the dark incubation. Wavelengths from both sides of the spectrum, from long (627 nm) to short wavelength (470-455 nm) had a stimulating effect on fumonisin biosynthesis compared to the dark incubation: fumonisins B(1) (FB(1)) and B(2) (FB(2)) production increased of about 40 fold under red, 35 fold under blue, 20 fold under royal blue, 10 fold under green, 5 fold under yellow and 3 fold under white light in comparison to the dark incubation. The transcriptional regulation of the FUM1 fumonisin biosynthesis gene was analyzed by Real time reverse transcriptase PCR quantification, revealing a correlation between fumonisin biosynthesis and gene expression. These findings show a role of light on the growth and the modulation of fumonisin biosynthesis and provide new information on the physiology of an important toxigenic maize pathogen.

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

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

Fusarium head blight (FHB) disease and deoxynivalenol (DON) contamination of wheat grains depend on multiple factors, above all climatic conditions, but also agronomic factors such as crop rotation, debris management, variety susceptibility and fungicide applications. Although it is generally believed that multiple strategies are more successful than a single strategy, only a few studies have shown the quantitative effect of combining multiple strategies.Field experiments have been conducted over three growing seasons in three sites in Northern Italy to evaluate the effect of previous crop residue management through tillage, variety susceptibility and triazole fungicide application on common wheat, according to a full factorial scheme. The following parameters were analyzed: FHB severity, grain yield and DON contamination.The collected data have clearly shown a close interaction between the factors involved in FHB severity and DON content, while the interactions were less significant for grain yield. In all nine trials, the DON contamination was significantly affected by the interaction of at least two of the compared factors, while the interaction between all three factors involved was significant in four trials. The most favourable scenario to avoid DON contamination (ploughing, moderately resistant variety, triazole application at heading) reduced the DON content by 97% compared to the worst one (direct sowing, susceptible variety, no fungicide application).Since the interaction between the agricultural practices have shown a synergistic effect, integrated multiple strategies, in areas characterized by a high risk of FHB, can be considered the very effective management means of reducing FHB and DON contamination in wheat.

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.

Biogas production represents one of the most economically attractive alternative technology for biofuel production from renewable resources. Generally, biogas plants are fed with agricultural residual products and food wastes, but the rising up of agricultural products contaminated by mycotoxins, such as maize silage not suitable for animal feeding, has pointed the question on the possibility to use this agricultural productfor biogas production. In this regards, a preliminary metagenomic analysis of microbial community residing in a mesophilic industrial-scale biogas fermenter, daily fed with contaminated maize silage, has been carried out to characterize the evolution of microbial community under the operating conditions and the mycotoxin content. Sample were collected from a biogas plant consisting of a three steps production taking place in a bioreactor, post-reactor and a storage tank. Total DNA was extracted from samples belonging to each steps of biogas production. Metagenomic analysis was carried out by analyzing the V4 variable region of bacterial and archaeal 16S rRNA gene. Mycotoxin content was analyzed in maize silage feeding the biogas plant and in the digestate from bioreactor, post-reactor and storage tank by immunoaffinity column clean-up (Myco6in1+®) and detected with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Over 3million high quality reads (about 1Gb) were generated on the Ion Torrent S5 Sequencing System. About 2.4 million reads were assigned for 16S analysis. In detail, metagenomic analysis revealed that Bacteria superkingdom was dominant (~96%) along the production steps, whereas Archaea were less represented (~4%). Within Bacteria the most abundant phylum was Firmicutes, mostly represented by Clostridia, followed by Bacteroidetes and Synergistetes. Within the superkingdom of Archaea, only microorganisms belonging to the phylum of Euryarchaeota were detected. Within Euryarchaeota the dominant genera were Methanosarcina and Methanoculleus. Chemical analysis on maize silage feeding the plants showed an initial mycotoxin contamination by DON (410 µg/kg), FB1 (3570 µg/kg), FB2 (810 µg/kg) and T-2 toxin (20 µg/kg), while AfB1, HT-2 Toxin, NIV, OTA and ZEA were not detected. After the first step of biogas production, a complete reduction of DON and T-2 content was achieved. These preliminary results suggest a possible absorption/degradation of mycotoxins in bioreactor tank and therefore further studies are needed to better elucidate the possible involvement of specific microbial taxa capable of mycotoxins reduction and the enzymatic pathways potentially involved in mycotoxin degradation.

Fusarium langsethiae, formally described as a new species over a decade ago, has been identified as the main producer of HT-2(HT2) and T-2 (T2) toxins in Europe in small cereal grains. Mycotoxin contamination caused by this Fusarium species can representa food safety hazard that deserves further attention. In the present work, the mycotoxin profile in wheat cultures of F.langsethiae is presented with particular reference to the production of major type-A trichothecenes and their glucosyl derivatives.F. langsethiae isolates, representative of the major Italian wheat cultivation areas, were tested for the production of T2,HT2, diacetoxyscirpenol (DAS) and neosolaniol (NEO), and relevant glucosyl derivatives. Liquid chromatography-tandem massspectrometry (LC-MS/MS) was used for the identification and chemical characterization of these metabolites. F. langsethiaeisolates under investigation resulted to be potent producers of T2, HT2 and NEO. Furthermore, a well-defined set of isolates,all originating from Central Italy, produced also DAS. All isolates were found to be able to produce HT2 glucosyl derivatives,whereas only traces of T2 glucoside were detected in one sample. Furthermore, two mono-glucosyl derivatives of NEO and onemono-glucoside derivative of DAS were identified and characterized. The screening for the presence/absence of glucosylatedtrichothecenes in analyzed fungal extracts revealed a general co-occurrence of these derivatives with the parent toxin at levelsthat could be roughly estimated to account up to 37% of the relevant unconjugated toxin. This is the first report of the productionof glucosylated trichothecenes by F. langsethiae cultured on small grains.

Surveys on the occurrence of type A trichothecenes in wheat, and particularly for the T-2 and HT-2 toxins, and information on the biology and epidemiology of the causative Fusarium species (i.e. F. langsethiae, F. sporotrichioides) are scarce in Italy, as compared to the more common type B trichothecene, deoxynivalenol and its producers. This 4-year monitoring of phytopathogenic Fusarium species on 183 seed lots of durum wheat shows wide distribution of F. langsethiae in Italy and the potential of several isolates of this fungus to produce high amounts of T-2 and HT-2 in wheat. Fusarium langsethiae was observed for approximately 48% of the analysed samples, with a maximum incidence for a single lot of 10.5%. Fusarium sporotrichioides was observed only in 2011, with an average incidence of 2% (range, 0-3%). A collection of F. langsethiae isolates representative of the main cultivation areas in Italy was established. These isolates showed great variability for their toxin production in vitro. Of 28 strains, all except one isolate can produce the T-2 and HT-2 toxins. HT-2 was generally in greater amounts than T-2, with an average concentration ratio for HT-2 to T-2 of 2.1 (range, 0.7-5.4). The artificial inoculation of wheat with three isolates of F. langsethiae produced no Fusarium head blight symptoms under field conditions. However, significantly higher incidence of F. langsethiae was seen on the kernels of inoculated plants, compared to the uninoculated controls.

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.

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

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.

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

Fusarium verticillioides and Fusarium proliferatum are the mainsource of fumonisins, a group of mycotoxins that can contaminate maize-based food and feed and cause diseases in humans and animals.We analyzed the effect of temperature (15-35°C), water activity(aw: 0.999-0.93), salinity (0-125 g/l NaCl), pH (5-8) and light ofdifferent wavelength (650-390 nm) on the growth, the productionof fumonisins B (FB) and the expression of FUM1 and FUM21. ForF. verticillioides the highest growth rate was measured at 25°C, awof 0.998-0.99, 0-25 g/l of NaCl and white light. Optimal conditionsfor fumonisin production were 30°C, aw of 0.99, 25 g/l of NaCl,pH 5, red and blue light. For Fusarium proliferatum the highestgrowth rate was measured at 25°C, aw of 0.99 and 0-25 g/l of NaCl,pH 7 and green light. Optimal conditions for fumonisin productionwere 25°C, aw of 0.998, 0 g/l of NaCl, pH 6, red and bluelight. F. verticillioides showed a better adaptability compared toF. proliferatum and was able to produce moderate levels of fumonisinsunder a wide range of conditions. FUM gene expression notalways mirrored FB production, indicating a post-transcriptionalmechanism regulationg fumonisin production. The study of theeffect of different environmental conditions on toxin productionshould provide information that can be used to develop strategiesto minimize the risk.

L'aflatossina M1 (AFM1) è il principale metabolita derivante dall'idrossilazionedell'aflatossina B1 (AFB1) presente nel latte di animali alimentati con mangimi contaminati da AFB1 ed è classificato nel gruppo 2B, potenzialmente cancerogeno per l'uomo, dall'AgenziaInternazionale per la Ricerca sul Cancro (IARC). Il livello limite della AFM1 nel latte crudo,trattato termicamente e destinato alla produzione di prodotti a base di latte è fissato a50ng/kg dal regolamento europeo numero 1881 del 2006. Essendo resistente ai comunitrattamenti dell'industria alimentare, la presenza di AFM1 è documentata in tutti i prodottidella filiera lattiero casearia, inclusi yogurt e formaggi, e rappresenta un serio pericolo perla salute. Lo sviluppo di metodi per la riduzione della contaminazione di aflatossine è untema cruciale e attuale, ed è complicato dalla necessità di preservare le qualità organolettichee nutrizionali della matrice trattata. In questo lavoro è stata valutata la capacità degradativadi una laccasi da Pleurotus eryngii verso l'AFM1, sia in buffer che in latte, ed il suo effettosulla componente proteica di questa matrice al fine di verificarne l'applicazione per ilmiglioramento della sicurezza di prodotti lattiero caseari. La riduzione di AFM1 in bufferdi sodio acetato pH 6.5 1mM, a 25°C, è di ca 50% dopo 1h di incubazione e risulta completadopo 72h. Simili risultati sono stati ottenuti in latte, sebbene la cinetica di degradazioneabbia registrato un rallentamento nelle prime tre ore di trattamento. L'analisi dei patternproteici in SDS-PAGE ha evidenziato una riduzione nell'intensità delle bande di ? e ?caseine, di ?-lattoglobulina e sieroalbumina bovina, contemporaneamente alla comparsadi aggregati proteici di peso molecolare superiore ai 200kDa. I dati presentati dimostranoil potenziale applicativo della laccasi per lo sviluppo di metodologie green di degradazionedi AFM1 in prodotti a base di latte e per applicazioni tecnologiche volte al miglioramentodella reologia e alla riduzione della componente allergenica in prodotti lattierocaseari.Parole chiave: sicurezza, latte, laccasi, aflatossina M1, cross-link di proteine, reologia dellatte, allergeni

Ochratoxin A (OTA) is a nephrotoxic and potentially carcinogenic mycotoxin produced by several species of Aspergillus and Penicillium, contaminating grapes, wine and a variety of food products.We recently isolated from OTA contaminated soil vineyard a novel free-livingstrain of Acinetobacter sp. neg1, ITEM 17016, able to degrade OTA into the nontoxic catabolic product OTalpha (OT?). Biochemical studies suggested that the degradation reaction proceeds via peptide bond hydrolysis with phenylalanine (Phe) release. In order to identify genes responsible for OTA degradationwe performed a differential gene expression analysis of ITEM 17016 grown in the presence or absence of the toxin. Among the differentially expressed genes, 6 peptidases up-regulated at 6 hours were identified. The degrading activity of the carboxypeptidase PJ_1540 was confirmed in vitro in a heterologous system. The enrichment analysis for Gene Ontology terms confirmed that OTA degradation proceeds through peptidase activities and revealed the over-representation of pathways related to Phe catabolism. These results indicate that Phe may represent an energy source for this Acinetobacter sp. neg1strain and that OTA degrading reaction triggers the modulation of further catabolic activities.

Fusarium equiseti and Fusarium semitectum represent the most abundant species in the Fusarium complex isolated from flowers, soybean pods and seeds in Argentina. The aim of the present study was to assess the production of major type A and type B trichothecenes (diacetoxyscirpenol, neosolaniol, T-2 toxin and HT-2 toxin, nivalenol, deoxynivalenol) and zearalenone by 40 F. equiseti and 22 F. semitectum isolates on rice culture. Mycotoxins were determined by HPLC with fluorescence detection after derivatisation with 1-anthronylnitrile for type A trichothecenes (i.e. diacetoxyscirpenol, neosolaniol, T-2 toxin and HT-2 toxin), by HPLC with UV detection for type B trichothecenes (i.e. nivalenol and deoxynivalenol), and by TLC for zearalenone. A total of 22 of 40 F. equiseti isolates produced diacetoxyscirpenol, nivalenol and ZEA alone or in combination, whereas only two of 20 F. semitectum isolates were nivalenol and ZEA producers. Both Fusarium species did not produce any deoxynivalenol, neosolaniol, T-2 toxin and HT-2 toxin. The variable retention in toxigenicity displayed by both fungal species suggests that these species have a saprophytic lifestyle in the soybean agroecosystem in Argentina.

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

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

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