Pathogenicity of Fumonisin-producing and Nonproducing Strains of Aspergillus Species in Section Nigri to Maize Ears and Seedlings.

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

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.

Effect of red thyme oil (Thymus vulgaris L.) vapours on fungal decay, quality parameters and shelf-life of oranges during cold storage.

This work has been aimed at studying the effect of red thyme oil (RTO, Thymus vulgaris L.) on the shelf-life and Penicillium decay of oranges during cold storage. RTO vapours significantly reduced (P ≤ 0.05) the percentage of infected wounds, the external growth area and the production of spores in inoculated orange fruit stored for 12 days at 7 °C in a polypropylene film selected for its appropriate permeability. Among the RTO compounds, p-cymene and thymol were the most abundant in packed boxes at the end of cold storage. The RTO vapours did not affect the main quality parameters of the oranges, or the taste and odour of the juice. The results have shown that an active packaging, using RTO vapours, could be employed, by the citrus industry, to extend the shelf-life of oranges for fresh market use and juice processing.

Genetic polymorphisms associated to SDHI fungicides resistance in selected Aspergillus flavus strains and relation with aflatoxin production.

Aspergillus flavus is a common and ubiquitous fungal species able to colonize several agricultural commodities, in both pre- and post-harvest conditions. This species represents a very harmful plant pathogen for its ability to synthesize aflatoxin B1, responsible for human primary hepatocellular carcinoma and classified as a group I (human carcinogenic) by the International Agency for Research on Cancer. Several approaches have been proposed to control A. flavus development and related aflatoxin production in field and storage conditions. The Succinate Dehydrogenase Inhibitor (SDHI) fungicide boscalid has been shown to control A. flavus growth and aflatoxin contamination both in vitro and in field experiments. However, this compound is classified as medium-high risk fungicide for triggering fungal resistance and, indeed, resistant strains can occur on crops treated with boscalid. In this paper, we selected laboratory A. flavus strains resistant to boscalid grown on agar medium containing 50 mg/L of boscalid. In order to investigate the molecular mechanism responsible for the resistant phenotype, specific primer pairs were designed to amplify the whole SdhB, SdhC and SdhD genes. By amino acid sequence analysis, two point mutations, Tyrosine replacing Histidine at codon 249 of SdhB (H249Y) and Arginine replacing Glycine at codon 91 of SdhC (G91R), were identified. The effect of SDHI boscalid and isopyrazam on mycelial growth and conidial germination was evaluated. Both resistant genotypes showed high resistance (MIC and EC50 > 1000 mg/L) to boscalid. A positive cross-resistance was found between boscalid and isopyrazam. Specific sub-lethal doses of both fungicides (0.5 mg/L of boscalid and 0.01 mg/L of isopyrazam) interfered with the mechanisms associated to pigmentation of colonies. In particular, fungal colonies appeared depigmented lacking the typical A. flavus green colour shown on un-amended fungicide medium. A strict correlation between lack of pigmentation and increasing aflatoxin production was also observed.

Influence of Lobesia botrana field control on black aspergilli rot and ochratoxin A contamination in grapes.

The grape berry moth Lobesia botrana is a key pest in vineyards in southern Europe. Damage caused by L. botrana larvae may encourage growth of black aspergilli, leading to ochratoxin A (OTA) accumulation in grapes. Field trials were conducted during three grape growing seasons (2005 through 2007) in Apulia, Italy, to evaluate an insecticide control strategy for L. botrana in the vineyard as an indirect method of reducing OTA contamination by reducing black aspergilli on the grapes. In the 2005 field trials, the insecticide treatment controlled attacks by L. botrana larvae and reduced OTA concentrations by up to 66% in the must samples of Negroamaro and Primitivo grape varieties. Significant differences (P < or = 0.05) also were observed in the incidence of black aspergilli. Environmental conditions in 2006 and 2007 resulted in a natural low level of infestation by L. botrana, low levels of OTA in both treated and untreated samples, and no significant differences between treated and nontreated samples. The results of our field study confirm previous reports that L. botrana is an important risk factor for OTA accumulation and are consistent with the hypothesis that controlling L. botrana in vineyards reduces OTA concentrations in grapes.

Genotyping by Amplified Fragment Length Polymorphism and malate metabolism performances of indigenous Oenococcus oeni strains isolated from Primitivo wine.

The Amplified Fragment Length Polymorphism (AFLP) technique was applied for the first time to investigate the genotyping of Oenococcus oeni, the most important species involved in malolactic fermentation (MLF) in wine. A total of 87 out of 220 lactic acid bacteria, isolates from "Primitivo" wine (Apulia, Italy) undergoing MLF, identified as O. oeni by species-specific PCR and 16S rRNA sequence analysis, were studied by AFLP analysis. Four main clusters were distinguished and three of them showed intraspecific homology higher than 60%. A total of 28 strains, representative of AFLP clusters, were tested for malate metabolism in order to gain information on their malolactic performances. Significant differences were observed among strains for malic acid consumed, biomass produced and specific malic acid consumption rate. These findings indicated that AFLP technique is reliable for typing O. oeni strains and that, together with metabolism studies it may be used to individuate possible candidates as industrial malolactic starters.

Cryptic subspecies and beauvericin production by Fusarium subglutinans from Europe.

Fusarium subglutinans is a maize ear rot pathogen and producer of beauvericin and other mycotoxins. This species has recently been split into two major phylogenetic within-species groups based on RFLP DNA sequence polymorphisms identified in the histone H3 and beta-tubulin sequences. A Pan European collection of the fungus originating mostly from maize was subjected to phylogenetic analysis by RFLP grouping and to chemical analysis for beauvericin production. Of the 62 isolates belonging to Group 1, 48 (77%) produced from 10 to 532 microg/g of beauvericin, whereas none of the 39 Group 2 isolates synthesized detectable amounts of the mycotoxin. The association between RFLP group and beauvericin production is consistent with the existence of two reproductively isolated subgroups within F. subglutinans and indicates that the toxicological risk of isolates of F. subglutinans depends on the group with which they are affiliated.

Further data on the production of beauvericin, enniatins and fusaproliferin and toxicity to Artemia salina by Fusarium species of Gibberella fujikuroi species complex.

The knowledge of toxigenic profiles of fungal plant pathogens is of extreme importance for evaluating the potential toxicity of infected plant products. Ninety-six fungal isolates belonging to 28 species in the Gibberella fujikuroi complex were studied for the production of beauvericin, enniatins and fusaproliferin in rice cultures. Toxin production ranged from 5 to 3000 microg/g for beauvericin, 2 to 131 microg/g for enniatins, and 4 to 440 microg/g for fusaproliferin. Beauvericin was the most common metabolite produced by 16 species followed by fusaproliferin with 11 species and enniatins with 4 species. The production of beauvericin by F. bulbicola, F. denticulatum, F. lactis, F. phyllophilum, F. pseudocircinatum, and F. succisae and fusaproliferin by F. antophilum, F. begoniae, F. bulbicola, F. circinatum, F. concentricum, F. succisae, and F. udum is reported here for the first time. Brine shrimp larvae were most sensitive to culture extracts of F. acutatum (up to 94+/-3%), F. concentricum (up to 99+/-1%), F. denticuatum (up to 100%) and F. sacchari (up to 100%). Toxicity towards brine shrimp was significantly correlated with the beauvericin content of the fungal extracts with few exceptions. These data indicate that beauvericin and fusaproliferin are common metabolites of species of the G. fujikuroi complex and pose a risk for a possible toxin accumulation in their respective host plant products. However, data from the brine shrimp bioassay showed that further toxic metabolites within this complex need to be characterized.

Biodiversity of complexes of mycotoxigenic fungal species associated with Fusarium ear rot of maize and Aspergillus rot of grape.

Fusarium ear rot of maize and Aspergillus rot of grape are two examples of important plant diseases caused by complexes of species of mycotoxigenic fungi. These complexes of species tend to be closely related, produce different classes of mycotoxins, and can induce disease under different environmental conditions. The infection of maize and grape with multiple fungal species and the resulting production of large classes of mycotoxins is an example of mutual aggressiveness of microorganisms toward host species as well as to humans and animals that eat feed or food derived from the infected and contaminated plants. Infection of crop plant with a complex of microbial species certainly represents a greater threat to a crop plant and to human and animal health than infection of the plant with a single fungal species.

European research on ochratoxin A in grapes and wine.

European wine production represents about 70% of world production and thus is an important export commodity. Ochratoxin A (OTA) was first detected as a wine contaminant in 1996 and the role of Aspergillus section Nigri and A. carbonarius in OTA production discovered in Europe in 1999. Subsequently Europe-wide surveys have shown that A. carbonarius is predominantly responsible for OTA contamination of grapes, wine and vine fruits. Analyses of wine samples throughout Europe have shown that there is a gradient in OTA concentration with a decrease from red, to rose and to white wines. The latitude of production is an important factor in determining risk from OTA contamination. Some geographic regions in Southern Europe are more prone to contamination with the toxigenic species and OTA. Ochratoxin A has also been found in much higher concentrations (max. 53 mug/kg) in dried vine fruit than in wine suggesting that A. carbonarius can dominate the drying vine fruit ecosystem. There is a significant lack of knowledge in Europe on conducive climatic conditions preharvest and their relationship with levels of risk from OTA contamination in grapes and their fate in wine production. This needs to be integrated with cultivation system to maximise the prevention of OTA entering this food chain.

Development of a quantitative real-time PCR assay for the detection of Aspergillus carbonarius in grapes.

Aspergillus carbonarius is the main species responsible for the production of ochratoxin A (OTA) in wine grapes. To monitor and quantify A. carbonarious in grapes, a quantitative real-time PCR assay was developed as a possible tool for predicting the potential ochratoxigenic risk. DNA extraction from grape berries was performed by using conventional extraction and clean up through EZNA Hi-bond spin columns. A TaqMan probe was used to quantify A. carbonarius genomic DNA in grape berries samples. An exogenous internal positive control was used to overcome DNA recovery losses due to matrix inhibition. The quantification of fungal genomic DNA in naturally contaminated grape was performed using the TaqMan signal versus spectrophotometrically measured DNA quantities (Log10) calibration curve with a linearity range from 50 to 5 x 10(-4) ng of DNA. A positive correlation (R2=0.92) was found between A. carbonarious DNA content and OTA concentration in naturally contaminated grape samples. This is the first application of TaqMan real-time PCR for identifying and quantifying A. carbonarius genomic DNA occurring in grapes. The rapid DNA extraction method for grapes, together with the commercial availability of reagents and instrumentation, allows to perform a remarkable number of reproducible assays (96-well format) in less than 4 h.

Effect of Lobesia botrana damages on black aspergilli rot and ochratoxin A content in grapes.

Sixty-nine wine grape samples of two varieties (Bombino Nero and Uva di Troia) were collected from four vineyards in a high ochratoxin A (OTA) incidence grape-growing area in Apulia, southern Italy, during the 2001 and 2002 crop harvests. The levels of toxin, measured in the two year harvests, ranged from 0.02 to 681 ng/g of fresh berries. In both surveys, higher levels of contamination by black aspergilli and OTA were found in both intact and rotten berries originating from bunches damaged by Lobesia botrana larvae as compared to bunches without L. botrana attacks. All berry samples with an OTA level >1 ng/g were contaminated by black aspergilli with a CFU>10(6), and all but one of these samples belonged to the Lobesia damaged berries group. This is the first evidence of an interaction between L. botrana damaged berries and OTA contamination, in field.

Characterization of Fusarium spp. isolates by PCR-RFLP analysis of the intergenic spacer region of the rRNA gene (rDNA).

In the present study, 44 Fusarium spp. isolates (5 Fusarium culmorum, 7 Fusarium graminearum, 1 Fusarium cerealis, 1 Fusarium poae, 26 Fusarium oxysporum, and 4 Gibberella fujikuroi species complex) were characterized morphologically, physiologically and genetically. All except one (Dutch Collection: CBS 620.72) were isolated from different hosts grown in various Spanish localizations. Morphological characterization was made according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). ZEA was determined by liquid chromatography and trichothecenes by gas chromatography. Confirmation was carried out by liquid chromatography-ion trap-mass spectrometry (ZEA) or gas chromatography-mass spectrometry (trichothecenes). Molecular characterization of isolates was performed using an optimized, simple and low-cost method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rRNA gene (rDNA). The results indicate that F. graminearum, F. culmorum and F. cerealis isolates were high ZEA and type B trichothecene producers, the F. poae isolate produced very low level of nivalenol while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. Restriction patterns of the IGS region did not show any relationship with the host, geographic origin of the isolate and mycotoxin-producing capacity. However, the haplotypes obtained with six restriction enzymes (CfoI, AluI, HapII, XhoI, EcoRI and PstI) permitted to discern the six assayed Fusarium species. Therefore, this is a rapid and suitable methodology that allows closely related strains to group and to estimate the genetic relationships between the groups.

Polymorphism and phylogenetic species delimitation in filamentous fungi from predominant mycobiota in withered grapes.

Filamentous fungi are the main pathogens of withered grapes destined for passito wine production. Knowledge of which species inhabit these post-harvest fruits and their pathogenicity is essential in order to develop strategies to control infection, but is still scarce. This study investigated the predominant mycobiota of withered grapes through a cultivation-dependent approach. Strain and species heterogeneity was evidenced on examining isolates collected over three consecutive years. Colony morphology and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis revealed the occurrence of several phenotypes and haplotypes, respectively. Strains were phylogenetically analyzed based on sequence typing of different genes or regions (e.g. calmodulin, ß-tubulin and internal transcribed spacer region). Beside the most common necrotrophic-saprophytic species of Penicillium, Aspergillus, Alternaria and Botrytis species responsible for fruit rot, other saprobic species were identified (e.g. Trichoderma atroviride, Sarocladium terricola, Arthrinium arundinis and Diaporthe eres) generally not associated with post-harvest fruit diseases. Species such as Penicillium ubiquetum, Cladosporium pseudocladosporioides, Lichtheimia ramosa, Sarocladium terricola, Diaporthe nobilis, Bipolaris secalis, Paraconiothyrium fuckelii and Galactomyces reessii that had never previously been isolated from grapevine or grape were also identified. Moreover, it was not possible to assign a species to some isolates, while some members of Didymosphaeriaceae and Didymellaceae remained unclassified even at genus level. This study provides insights into the diversity of the epiphytic fungi inhabiting withered grapes and evidences the importance of their identification to understand the causes of fruit diseases. Finally, phylogenetic species delimitation furnished data of interest to fungal taxonomy.

An integrated taxonomic study of Fusarium langsethiae, Fusarium poae and Fusarium sporotrichioides based on the use of composite datasets.

An integrated systematic study was carried out to clarify the taxonomical position and relationship of Fusarium langsethiae to other taxa within the Fusarium section Sporotrichiella. Strains of this species were compared with strains of the closely related species Fusarium poae and Fusarium sporotrichioides using a composite dataset. This set consisted of DNA sequences derived from the ribosomal internal transcribed spacer (ITS) regions, partial sequences of the ribosomal intergenic spacer (IGS) region, the beta-tubulin and translation elongation factor-1 alpha (EF-1alpha) genes, AFLP fingerprints, chromatographic data on secondary metabolites and morphological data and growth characteristics. From these combined data, a consensus matrix was calculated by taking the mean of all pairwise distances between single isolates over all separate datasets. The consensus matrix was used as the basis for the construction of a UPGMA dendrogram and a multidimensional scaling, both of which revealed a clear separation of the three taxa. Partial IGS, EF-1alpha and beta-tubulin sequence-as well as chromatography-and AFLP-derived similarities turned out to be comparably consistent, while ITS sequence- and morphology-derived similarity matrices were rather divergent.

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.

Aspergillus section Nigri as contributor of fumonisin B(2) contamination in maize.

Fumonisins (FBs), which are carcinogenic mycotoxins, are known to be typically produced by several phytopathogenic fungal species belonging to the genus Fusarium. F. proliferatum and F. verticillioides, two important pathogens of maize worldwide, 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, FB2 and FB4). In a survey on maize carried out in Central Italy, 17 maize kernel samples were collected at harvest and analysed for FB1, FB2 and FB3, as well as fungal contamination, with a particular attention to the species-producing FBs. All 17 samples 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% of kernels. 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 µg g(-1), whereas FB2 ranged from 0.04 to 13.2 µg g(-1). The ratio of FB2/FB1 contamination in the maize samples was evaluated and the highest values occurred in samples contaminated with Aspergillus section Nigri. Thirty strains of Aspergillus section Nigri isolated from these samples were molecularly 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 µg g(-1). This is the first report showing the capability of Aspergillus section Nigri from maize to produce FB2 and its possibility to contribute to FB accumulation in kernels.

Molecular biodiversity of mycotoxigenic fungi that threaten food safety.

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

Phylogenetic characterization and ochratoxin A--fumonisin profile of black Aspergillus isolated from grapes in Argentina.

Aspergillus section Nigri populations isolated from seven growing regions from Argentina were characterized by sequencing in order to identify species responsible for production of ochratoxin A (OTA) and fumonisins (FB(s)). Sequences of genes encoding calmodulin, ß-tubulin, the second largest subunit of RNA polymerase II and translation elongation factor 1 alpha were analysed. The phylogenetic analysis showed the presence of six lineages: A. carbonarius, A. tubingensis, A. niger, A. japonicus, A. homomorphus and A. foetidus grouped in four major clusters. The molecular tools used allowed the identification for the first time of A. homomorphus from vineyards. OTA production confirmed the importance of A. carbonarius as the main ochratoxigenic species isolated and, to a variable degree, of A. niger and A. tubingensis, which were by far the most commonly occurring species on grapes in Argentina. The only strains able to produce OTA and fumonisins (B(2)-B(4)) belong to the A. niger cluster.