Transcriptional regulation of enniatins production by Fusarium avenaceum.

AIMS: The aim of this study was to analyse the transcriptional regulation of enniatins (ENs) production in Fusarium avenaceum. METHODS AND RESULTS: We develop a new method to quantify ENs in FDM agar medium. We performed an LC/MS/MS analysis to evaluate enniatin A, A1, B, B1 and B4 production by seven F. avenaceum strains and, in a time-course experiment, by ITEM 3404 to analyse the transcriptional regulation of the esyn1 gene. The expression profile, achieved by Real time reverse transcriptase assay, showed an activation of gene transcription at the seventh day of incubation, corresponding to the higher increase of total ENs production. Enniatin B was the most abundant ENs analogues, representing the 90% of total ENs. The relative percentage of ENs remained unaltered during the experiment. CONCLUSIONS: We reported a transcriptional regulation of esyn1 responsible for the modulation of ENs biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Enniatins are cyclic depsipeptides metabolites with a wide range of biological activities. They are also widespread contaminants in grains and cereals due to infection by enniatin-producing Fusarium species. This is the first article describing the transcriptional regulation of esyn1 gene that modulates ENs production in Fusarium avenaceum and provides new knowledge about the molecular mechanism underlying the biosynthesis of these important fungal metabolites in this toxigenic fungal species.

Searching for genes responsible for patulin degradation in a biocontrol yeast provides insight into the basis for resistance to this mycotoxin.

Patulin is a mycotoxin that contaminates pome fruits and derived products worldwide. Basidiomycete yeasts belonging to the subphylum Pucciniomycotina have been identified to have the ability to degrade this molecule efficiently and have been explored through different approaches to understand this degradation process. In this study, Sporobolomyces sp. strain IAM 13481 was found to be able to degrade patulin to form two different breakdown products, desoxypatulinic acid and (Z)-ascladiol. To gain insight into the genetic basis of tolerance and degradation of patulin, more than 3,000 transfer DNA (T-DNA) insertional mutants were generated in strain IAM 13481 and screened for the inability to degrade patulin using a bioassay based on the sensitivity of Escherichia coli to patulin. Thirteen mutants showing reduced growth in the presence of patulin were isolated and further characterized. Genes disrupted in patulin-sensitive mutants included homologs of Saccharomyces cerevisiae YCK2, PAC2, DAL5, and VPS8. The patulin-sensitive mutants also exhibited hypersensitivity to reactive oxygen species as well as genotoxic and cell wall-destabilizing agents, suggesting that the inactivated genes are essential for tolerating and overcoming the initial toxicity of patulin. These results support a model whereby patulin degradation occurs through a multistep process that includes an initial tolerance to patulin that utilizes processes common to other external stresses, followed by two separate pathways for degradation.

Natural occurrence of fumonisin B2 in red wine from Italy.

The potential risk of exposure to fumonisin B(2) (FB(2)) in the grape-wine chain has recently been revealed after a report of Aspergillus niger in grapes and its ability to produce FB(2) and FB(4). The occurrence of these two fumonisins in wine was investigated by LC/MS/MS in 51 market samples (45 red, five white and one rose wine) produced in various Italian regions. Nine samples of red wine were found to be contaminated by fumonisin B(2) at levels ranging from 0.4 to 2.4 ng/ml, while FB(4) was not detected in any of the tested samples. This is the first report on the natural occurrence of FB(2) in wine, indicating that, although at low levels, there is a potential risk of FB(2) exposure for the wine-consumer.

Fumonisin B(2) production by Aspergillus niger from grapes and natural occurrence in must.

Aspergillus niger has been recently found to produce fumonisin B(2) (FB(2)). Thirty-one strains belonging to four Aspergillus species isolated from grape were evaluated for FB(2) production on agar plates. Four out of eight strains of A. niger produced FB(2) (29-293 microg g(-1)). None of the strains of A. uvarum (n = 7), A. tubingensis (8) and A. carbonarius (8) produced detectable amounts of toxin. The capability to produce FB(2) was also confirmed by some A. niger strains artificially inoculated on grape berries. Natural occurrence of FB(2), at levels of 0.01 and 0.4 microg ml(-1), was found in two samples of must collected in Apulian cellars in 2007. This is the first report of FB(2) contamination in must. These findings suggest that there is a potential risk of exposure to FB(2) in the grape-wine chain for consumers and that A. niger may represent the major fumonisin-producing species among black Aspergilli occurring on grapes.

Dominance of Group 2 and fusaproliferin production by Fusarium subglutinans from Iowa maize.

Fusarium subglutinans (teleomorph Gibberella subglutinans, member of the Gibberella fujikuroi complex) is an important toxigenic pathogen of maize. Recently, two cryptic species (Groups 1 and 2) have been described within F. subglutinans, but little is known about the occurrence of the two groups in North America or their relative capacities to produce mycotoxins. In this study, 58 F. subglutinans strains from kernels of maize grown in Iowa, USA, were evaluated for cryptic speciation and production of the mycotoxins fusaproliferin and beauvericin. Fifty-six of the 58 strains (97%) belonged to Group 2, and two strains belonged to Group 1, based on restricted fragment length polymorphisms derived from amplification of histone H3 and beta-tubulin gene fragments. Fifty-four Group 2 strains and both Group 1 strains produced fusaproliferin at concentrations ranging from 12 to 3000 microg g(-1) of solid maize culture. None of the F. subglutinans strains from Iowa produced beauvericin at detectable amounts, although most F. subglutinans strains from Europe and elsewhere are beauvericin producers. These results indicate that F. subglutinans strains infecting maize kernels in Iowa belong almost exclusively to Group 2 and that they have a high potential for fusaproliferin production; furthermore, the results confirm an association between Group 2 genotypes and lack of beauvericin production. This is the first report characterizing the phylogenetic groups of F. subglutinans occurring in Iowa; the predominance of Group 2 suggests that populations of the fungus in Iowa and Europe remain isolated from each other. Fusaproliferin contamination of grain appears to be a risk wherever F. subglutinans occurs, but beauvericin contamination from F. subglutinans is associated only with Group 1.

Factors affecting the production of Trichoderma harzianum secondary metabolites during the interaction with different plant pathogens.

AIMS: Strains of Trichoderma spp. produce numerous bioactive secondary metabolites. The in vitro production and antibiotic activities of the major compounds synthesized by Trichoderma harzianum strains T22 and T39 against Leptosphaeria maculans, Phytophthora cinnamomi and Botrytis cinerea were evaluated. Moreover, the eliciting effect of viable or nonviable biomasses of Rhizoctonia solani, Pythium ultimum or B. cinerea on the in vitro production of these metabolites was also investigated. METHODS AND RESULTS: T22azaphilone, 1-hydroxy-3-methyl-anthraquinone, 1,8-dihydroxy-3-methyl-anthraquinone, T39butenolide, harzianolide, harzianopyridone were purified, characterized and used as standards. In antifungal assays, T22azaphilone and harzianopyridone inhibited the growth of the pathogens tested even at low doses (1-10 microg per plug), while high concentrations of T39butenolide and harzianolide were needed (>100 microg per plug) for inhibition. The in vitro accumulation of these metabolites was quantified by LC/MS. T22azaphilone production was not enhanced by the presence of the tested pathogens, despite its antibiotic activity. On the other hand, the anthraquinones, which showed no pathogen inhibition, were stimulated by the presence of P. ultimum. The production of T39butenolide was significantly enhanced by co-cultivation with R. solani or B. cinerea. Similarly, viable and nonviable biomasses of R. solani or B. cinerea increased the accumulation of harzianopyridone. Finally, harzianolide was not detected in any of the interactions examined. CONCLUSIONS: The secondary metabolites analysed in this study showed different levels of antibiotic activity. Their production in vitro varied in relation to: (i) the specific compound; (ii) the phytopathogen used for the elicitation; (iii) the viability of the elicitor; and (iv) the balance between elicited biosynthesis and biotransformation rates. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of cultures of phytopathogens to enhance yields of Trichoderma metabolites could improve the production and application of novel biopesticides and biofertilizers based on the active compounds instead of the living microbe. This could have a significant beneficial impact on the management of diseases in crop plants.

Simultaneous determination of aflatoxin B1 and ochratoxin A and their natural occurrence in Mediterranean virgin olive oil.

Olive oil, the most important dietary fat source of the Mediterranean diet, can be contaminated by mycotoxins. An efficient analytical method for the simultaneous determination of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in olive oil is reported. Thirty commercial samples of virgin olive oil, purchased in olive-press plants and supermarkets in southern Italy and North Africa, were analysed to verify the analytical procedure and monitor mycotoxin contamination. A simple, rapid and economic method was set up and tested for both the extractive step and the clean-up procedures for simultaneous AFB1 and OTA determination in olive oil. Data obtained showed that OTA was detected with high frequency (80%) in samples from both geographical areas (up to 17.0 ng g-1), while AFB1 was found from three of four samples from North Africa (up to 2.4 ng g-1). In addition, 'not labelled' oil samples proved to be more contaminated by OTA then 'labelled' samples (mean values of 2.47 and 0.66 ng g-1, respectively). These findings indicate that olive oil can be significantly contaminated by mycotoxins and confirm that a scrupulous application of European Regulation 1019/2002 (European Commission 2002), which prohibits the sale of non-labelled olive oil, is strongly recommended. Conventional qualitative parameters such as peroxide number, spectrophotometric evaluation and acid values were not correlated with mycotoxin occurrence.

Effect of domestic cooking on human bioavailability of naringenin, chlorogenic acid, lycopene and beta-carotene in cherry tomatoes.

BACKGROUND: Epidemiological data showed that tomato and tomato product (sauce, paste) consumption is associated with a protective effect against the development of some chronic-degenerative diseases. Tomato antioxidant bioactive molecules such as carotenoids and polyphenols could be responsible, at least in part, for the healthy effect observed. The bioavailability of these compounds is an essential requirement to sustain their in vivo role. While it is well known that many factors can influence the bioaccessibility of carotenoids from the food matrix, there is little information about the factors affecting phenolic compounds' bioaccessibility. AIM OF THE STUDY: This investigation was carried out to evaluate the effect of domestic cooking on the bioavailability in humans of antioxidant molecules after the administration of a test meal containing cherry tomatoes. METHODS: A cross-over design was conducted. Subjects (3 females and 2 males) consumed experimental meals containing fresh and cooked cherry tomatoes. Blood collection was performed at different time intervals (0, 2, 4, 6, 8 and 24 h). RESULTS: Carotenoid and phenol plasma concentrations were measured. Plasma levels of lycopene and beta-carotene were not significantly different with respect to the baseline after ingestion of both the test meals, while plasma concentrations of naringenin and chlorogenic acid increased significantly with respect to the baseline (P<0.05) after administration of cooked cherry tomatoes, but not after administration of fresh cherry tomatoes. CONCLUSIONS: The present study indicated that domestically cooked tomatoes significantly increase naringenin and chlorogenic acid plasma levels. Considering that both naringenin and chlorogenic acid are widely studied for their potential healthy properties, evidence of their bioavailability and of the factors influencing their bioaccessibility is an important tool to sustain the possibility that these polyphenols play a biological role in human physiology.

Influence of antioxidants in virgin olive oil on the formation of heterocyclic amines in fried beefburgers.

An association between the intake of heterocyclic amines (HAs) and the development of cancer has been observed in some epidemiological studies, while in other studies no such correlation has been found. HAs are mutagenic/carcinogenic compounds formed at low levels via the Maillard reaction and a free radical mechanism during cooking of animal tissue. The addition of pure antioxidants or foods containing antioxidants has previously been shown to decrease the amount of HAs formed during cooking. In this study, beefburgers were fried in six different oils: refined olive oil, virgin olive oil, virgin olive oil depleted of phenols, rapeseed oil, virgin olive oil with rosemary extract and refined olive oil with rosemary extract. The content of antioxidative compounds in the virgin olive oil and the rosemary extract was determined. The beefburgers were analysed with regards to 12 different HAs by solid phase extraction and HPLC analysis. MeIQx, 4,8-DiMeIQx, PhIP, Harman and Norharman were detected in all beefburgers fried in the different oils, but the relative amounts varied. Frying in virgin olive oil reduced the formation of HAs compared with refined olive oil. This effect is probably due to the content of phenols in the virgin olive oil. The HA-reducing effect of virgin olive oil decreased during storage, but the addition of rosemary extract may prevent this decrease.

Occurrence of beauvericin and enniatins in wheat affected by Fusarium avenaceum head blight.

We evaluated Fusarium contamination and the levels of hexadepsipeptide mycotoxins in 13 wheat samples affected by head blight in Finland. Fusarium avenaceum was the dominant species (91%) isolated from all samples, but isolates of F. culmorum (4%), F. tricinctum (3%), and F. poae (2%) also were recovered. Beauvericin (0.64 to 3.5 microg/g) was detected in all 13 samples. Enniatin B (trace to 4.8 microg/g) was detected in 12 samples, enniatin B(1) (trace to 1.9 microg/g) was detected in 8 samples, and enniatin A(1) (trace to 6.9 microg/g) was detected in 10 samples. Ten of 13 strains of F. avenaceum and 2 strains of F. poae and F. tricinctum produced beauvericin in culture on rice (trace to 70, 9.4, and 33 microg/g, respectively). All strains also produced enniatins (trace to 2,700 microg/g). This is the first report on the natural co-occurence of beauvericin and enniatins in wheat infected predominantly by F. avenaceum.

Analysis of bacterial lipodepsipeptides by matrix-assisted laser desorption/ionisation time-of-flight and high-performance liquid chromatography with electrospray mass spectrometry.

Strains of certain plant pathogenic bacteria, in particular several pathovars of Pseudomonas syringae, are known to produce cyclic lipodepsipeptides (LDPs) endowed with peculiar structural features and noticeable biological activities. In this study, a mass spectrometry procedure is proposed for screening LDP-producing bacterial strains and for identifying and assessing individual LDPs. After matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) screening of thirteen P. syringae strains for LDP production, the extracts from culture filtrates of eight positive strains were subjected to electrospray mass spectrometry for the identification of LDPs. Five strains were found to produce two forms of syringomycins (SR-E and SR-G) and two forms of syringopeptin 25 (SP25A and SP25B); two strains produced SR-E, SR-G and a new form of SP22; one strain produced syringotoxin (ST) and syringostatin A (SS-A) in addition to SP25A and SP25B. The yield in culture of two major LPDs: SR-G (3.2-13.8 mg x L(-1)) and SP25A (41.6-231.5 mg x L(-1)) was assessed by and high-performance liquid chromatography with electrospray mass spectrometry (HPLC/ESI-MS) in both scan and single ion monitoring (SIM) modes. Results of this investigation showed that the mass spectrometry protocol developed here is a precise and reliable method for screening bacterial strains for LDP production and for assessing the amount of each metabolite under various culture conditions. This could be of practical value in view of potential applications, e.g. biocontrol of post-harvest fungal diseases.

Simultaneous determination of beauvericin, enniatins, and fusaproliferin by high performance liquid chromatography.

A rapid, sensitive and inexpensive HPLC method for routine screening of beauvericin, fusaproliferin, and enniatin B(1), A(1), and B has been optimized. Detection limits were determined, ranging between 0. 5 and 3.6 ng according to the compound obtained after spiking samples with each mycotoxin at 10-56 microg/mL concentration range; recoveries averaging from 56 to 74% were obtained. LC-MS conditions for enniatin analyses by API electrospray technique were set up, this allowing a unique identification of three different enniatins.