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Mycoremediation is an important process that targets the removal of petroleum hydrocarbons by fungi. Fungi have advantages with their extensive enzymatic systems, rapid adaptation to toxic organic pollutants, and to adverse environmental conditions. In this study, the colorimetric method was used for the preliminary investigation of petroleum degradation with ten fungal strains. Petroleum degradation ability of spore suspension, live biomass (fungal pellet and disc) and cell-free culture supernatant of the potent A. ochraceus strain were investigated by gravimetric analysis. It was found that the fungal disc (94%) was more successful than the spore suspension (87%) in petroleum degradation under physiological conditions determined as pH:5.0, 1% of petroleum concentration, 5% (v/v) of inoculum concentration (with spore suspension) and 1 g/100 mL of inoculum amount (with fungal disc) and 7 days of the incubation period. The degradation rate constant and half-life period of spore suspension were calculated as 0.291 day−1 and t1/2 = 0.340 and of the fungal disc were 0.401 day−1 and t1/2 = 0.247. Although, 7.5% and 10% (v/v) concentration of cell-free culture supernatant achieved more than 80% petroleum removal, it was not as effective as a fungal disc. According to gas chromatography/mass spectrometry analysis, the fungal disc of A. ochraceus strain degraded long-chain n-alkanes such as C35 and C36 more effectively than n-alkanes in the range of C22–C34. The fact that the A. ochraceus NRRL 3174 strain has a high petroleum degradation capacity as well as being a potent biosurfactant producer will provide a different perspective to advanced mycoremediation studies. [ABSTRACT FROM AUTHOR]

Aspergillus ochraceus is a soil fungus known to produce ochratoxin A, a harmful secondary metabolite. Prevention and control of fungal pathogens mostly rely on chemical fungicides, which is one of the contributing factors in the emergence of the fungal resistance, hence novel methods for fungal eradication have been extensively researched. The cold atmospheric pressure (CAP) plasma generated in ambient air has been recently applied in microbial decontamination. Here we used the diffuse coplanar surface barrier discharge in inactivation of a toxigenic strain A. ochraceus. The plasma-treated conidia and mycelium exhibited morphological changes such as ruptures and desiccation. Mycelium dehydration and changes in the chemical composition of hyphal surface accompanied plasma treatment. The growth of 26 h old mycelia were significantly restricted after 30 s of plasma treatment. The conidial vitality declined 4 logs after 180 s of plasma exposure leading to almost complete decontamination. After shorter plasma treatment of conidia, the ochratoxin A (OTA) production increased at the early stage of cultivation, but the overall level was significantly reduced compared to untreated samples after longer cultivation. Our results indicated that the fungal growth and the OTA production were significantly changed by plasma treatment and underscored CAP plasma as a promising method in the decontamination of A. ochraceus without a risk to generate strains with increased OTA production. [ABSTRACT FROM AUTHOR]

Key message: OTA-producing strain Aspergillus ochraceus induc ed necrotic lesions, ROS accumulation and defense responses in Arabidopsis . Primary metabolic and defense-related proteins changed in proteomics. Ascorbate-glutathione cycle and voltage-dependent anion-selective channel proteins fluctuated. Abstract: Mycotoxigenic fungi, as widespread contaminants by synthesizing mycotoxins in pre-/post-harvest infected plants and even stored commercial cereals, could usually induce plant-fungi defense responses. Notably, ochratoxin A (OTA) is a nephrotoxic, hepatotoxic, teratogenic, immunotoxic and phytotoxic mycotoxin. Herein, defense responses of model system Arabidopsis thaliana detached leaves to infection of Aspergillus ochraceus 3.4412, an OTA high-producing strain, were studied from physiological, proteomic and transcriptional perspectives. During the first 72 h after inoculation (hai), the newly formed hypersensitive responses-like lesions, decreased chlorophyll content, accumulated reactive oxygen species and upregulated defense genes expressions indicated the defense response was induced in the leaves with the possible earlier motivated jasmonic acid/ethylene signaling pathways and the later salicylic acid-related pathway. Moreover, proteomics using two-dimensional gel electrophoresis 72 hai showed 16 spots with significantly changed abundance and 13 spots corresponding to 12 unique proteins were successfully identified by MALDI-TOF/TOF MS/MS. Of these, six proteins were involved in basic metabolism and four in defense-related processes, which included glutathione- S-transferase F7, voltage-dependent anion-selective channel protein 3 (VDAC-3), osmotin-like protein OSM34 and blue copper-binding protein. Verified from proteomic and/or transcriptional perspectives, it is concluded that the primary metabolic pathways were suppressed with the ascorbate-glutathione cycle fluctuated in response to A. ochraceus and the modulation of VDACs suggested the possibility of structural damage and dysfunction of mitochondria in the process. Taken together, these findings exhibited a dynamic overview of the defense responses of A. thaliana to A. ochraceus and provided a better insight into the pathogen-resistance mechanisms in plants. [ABSTRACT FROM AUTHOR]

The biotransformation of hydrocortisone ( 1) by Fusarium solani and Aspergillus ochraceus was investigated for the first time. After 10 days at 30 °C, just one metabolite was produced by both fungi: 11β, 17α, 20β, 21-tetrahydroxypregn-4-en-3-one ( 2) established on the basis of spectroscopic data. The reaction was reduction of the 20-carbonyl group. Time course study determined by HPLC showed 60 and 45 % yield for the metabolite by F. solani and A. ochraceus, respectively. [ABSTRACT FROM AUTHOR]

Production of multiple xylanases, in which each enzyme has a specific characteristic, can be one strategy to achieve the effective hydrolysis of xylan. Three xylanases (xyl 1, xyl 2, and xyl 3) from Aspergillus ochraceus were purified by chromatography using diethylaminoethyl (DEAE) cellulose, Biogel P-60, and Sephadex G-100 columns. These enzymes are glycoproteins of low molecular weight with an optimum temperature at 60 °C. The glycosylation presented is apparently not related to thermostability, since xyl 3 (20 % carbohydrate) was more thermostable than xyl 2 (67 % carbohydrate). Xyl 3 was able to retain most of its activity in a wide range of pH (3.5-8.0), while xyl 1 and xyl 2 presented optimum pH of 6.0. Xyl 1 and xyl 2 were activated by 5 and 10 mM MnCl and CoCl, while xyl 3 was activated by 1 mM of the same compounds. Interestingly, xyl 2 presented high tolerance toward mercury ion. Xylanases from A. ochraceus hydrolyzed xylans of different origins, such as birchwood, oat spelt, larchwood, and eucalyptus (around 90 % or more), except xyl 2 and xyl 3 that hydrolyzed with lesser efficiency eucalyptus (66.7 %) and oat spelt (44.8 %) xylans. [ABSTRACT FROM AUTHOR]

Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular β-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. β-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 °C and 3.0-5.5, respectively. β-xylosidase was stable in acidic pH (3.0-6.0) and 70 °C for 1 h. The enzyme was activated by 5 mM MnCl (28 %) and MgCl (20 %) salts. The β-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-β- d-xylopyranoside, exhibiting apparent K and V values of 0.66 mM and 39 U (mg protein) respectively, and to a lesser extent p-nitrophenyl-β- d-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel β- d-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by β-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose. [ABSTRACT FROM AUTHOR]

Among the various factors correlated with toxin production in fungi, oxidative stress is a crucial one. In relation to this, an important role is played by oxidative stress-related receptors. These receptors can transduce the 'oxidative message' to the nucleus and promote a transcriptional change targeted at restoring the correct redox balance in the cell. In Aspergillus parasiticus, the knockout of the Ap yapA gene, a homologue of the yeast Yap-1, disables the fungus's capacity to restore the correct redox balance in the cell. As a consequence, the onset of secondary metabolism and aflatoxins synthesis is triggered. Some clues as to the involvement of oxidative stress in the regulation of ochratoxin A (OTA) synthesis in Aspergillus ochraceus have already been provided by the disruption of the oxylipin-producer Ao loxA gene. In this paper, we add further evidence that oxidative stress is also involved in the regulation of OTA biosynthesis in A. ochraceus. In fact, the use of certain oxidants and, especially, the deletion of the yap1-homologue Ao yap1 further emphasize the role played by this stress in controlling metabolic and morphological changes in A. ochraceus. [ABSTRACT FROM AUTHOR]

Delay between harvesting of coffee berries and onset of processing are common in most of the coffee-producing countries worldwide. Delay in processing is considered to be a negative operation in coffee production with respect to quality and safety. The aim of this study was to reconfirm the impact of delay in processing on ochratoxin A contamination in coffee and subsequent coffee quality. Delay in processing was found to favor higher mold incidence in cherry as compared to parchment coffee of both arabica and robusta. The incidence of Aspergillus ochraceus in cherry coffee was double compared to the rate of parchment coffee. No definite correlation was found between ochratoxin A contamination and delay in processing in cherry and parchment. Delay in processing increased the drying rate in cherry preparation as compared to parchment. Among the coffee types, delay in processing reduced the drying time in robusta when compared to arabica coffee. In cherry, at least 1 day was reduced in arabica, while in robusta cherry the drying days reduced by 5 days. In parchment coffee, processing delay was found to increase drying days by at least one in both arabica and robusta. Delay in processing had a negative effect on cup quality in both coffee types and processing methods. The present study confirms that delays between harvesting the coffee cherries and onset of processing increases the risk of ochratoxigenic mold and ochratoxin A contamination in coffee, together with poor cup quality. [ABSTRACT FROM AUTHOR]

Genome sequencing for many important fungi has begun during recent years; however, there is still some deficiency in proteome profiling of aspergilli. To obtain a comprehensive overview of proteins and their expression, a proteomic approach based on 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry was used to investigate A. ochraceus. The cell walls of fungi are exceptionally resistant to destruction, therefore two lysis protocols were tested: (1) lysis via manual grinding using liquid nitrogen, and (2) mechanical lysis via rapid agitation with glass beads using MagNalyser. Mechanical grinding with mortar and pestle using liquid nitrogen was found to be a more efficient extraction method for our purpose, resulting in extracts with higher protein content and a clear band pattern in SDS-PAGE. Two-dimensional electrophoresis gave a complex spot pattern comprising proteins of a broad range of isoelectric points and molecular masses. The most abundant spots were subjected to mass spectrometric analysis. We could identify 31 spots representing 26 proteins, most of them involved in metabolic processes and response to stress. Seventeen spots were identified by de novo sequencing due to a lack of DNA and protein database sequences of A. ochraceus. The proteins identified in our study have been reported for the first time in A. ochraceus and this represents the first proteomic approach with identification of major proteins, when the fungus was grown under submerged culture. [ABSTRACT FROM AUTHOR]

A number of polyketide synthase gene sequences from Aspergillus ochraceus were isolated by both SSH-PCR and degenerate PCR. The deduced amino acid sequences of the corresponding cloned pks DNA fragments were then aligned with the amino acid sequences of other polyketide synthase enzymes. One of these pks genes is essential for ochratoxin A biosynthesis (OTA-PKS). The OTA-PKS was most similar to methylsalicylic acid synthase (MSAS) type PKS proteins based on the alignment of the ketosynthase domains while if the acyl transferase domains were aligned it appeared to be more similar to PKS enzymes from Cochliobolus heterostrophus. The three PKS proteins identified by degenerate PCR were all from different PKS types, one was a MSAS type enzyme, the second was similar to the PKS proteins involved in lovastatin biosynthesis while the third was not similar to any of the other phylogenetic groupings. Data is presented which suggests that the use of phylogenetic analysis to predict the function of PKS proteins/genes is likely to be significantly enhanced by analyzing more than one domain of the protein. [ABSTRACT FROM AUTHOR]

The aim of this work was to evaluate the incidence of potential ochratoxigenic mycoflora and ochratoxin A (OA) in poultry, pig and rabbit feeds. Eighty poultry, pig and rabbit feed samples were taken at random from factories located from Córdoba province, Argentina, over a period of 8 months. Isolation and quantitative enumeration of fungal propagules were done on DRBC and DG18 media. The predominant species were A. candidus, A. flavus, A. terreus, A. parasiticus, P. implicatum P. minioluteum, P. crustosum and P. citrionigrum. The distribution of section Nigri species varied according to the feedstuffs analysed. The frequency of A. niger var. niger was noticeably high in poultry feed samples on DRBC medium. The Nigri section species was present at moderate mean colony counts (CFU/g) from three feeds. Mycotoxin analysis of these samples showed that OA was detected in 15%, 10% and 12% of pig, poultry and rabbit feed samples, respectively. The mean levels detected ranged between 15 and 25 ng/g from three feeds. The presence of ochratoxigenic species of Nigri section and OA in feeds indicates the risk of potential exposure of poultry, pigs and rabbits through the ingestion of feeds. [ABSTRACT FROM AUTHOR]

Mycotoxic nephropathy was induced in 18 young pigs by diets contaminated with strains of Aspergillus ochraceus containing ochratoxin A (OTA) and penicillic acid (PA) at levels corresponding to those naturally encountered in animal feeds in Bulgaria. Haematological and biochemical parameters, as well as the morphological and ultrastructural changes in various internal organs, and especially in the kidneys, were examined at different stages of development of the disease. A mottled surface of the kidneys was only seen in pigs exposed to a mouldy diet containing 180 ppb OTA for 3 months, but microscopic lesions, as well as changes in various haematological and biochemical parameters, were observed in all groups exposed to the same mouldy diet containing only 90 or 180 ppb OTA. Histological examination showed two types of change: degenerative changes affecting the epithelial cells of the proximal tubules, which predominated at the initial stage, and proliferative changes in the interstitium, which predominated at the later stage of the disease. Telangiectasis and lymph stasis were also seen, as well as degenerative changes in the capillary endothelium. The characteristic renal lesions were similar to those observed in spontaneous cases of mycotoxic porcine nephropathy in Bulgaria, but they were a little different from the classic Danish porcine nephropathy. The enhanced toxicity of OTA in our study may be due to a synergistic effect between OTA and PA or to some other unknown metabolites produced by the same ochratoxinogenic strains of A. ochraceus. [ABSTRACT FROM AUTHOR]

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [H]acetate, N-acetyl- D-[H]glucosamine and D-[C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens. [ABSTRACT FROM AUTHOR]

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The heat stability of ochratoxin A in green coffee beans inoculated with Aspergillus ochraceus was studied. Heat treatment (roasting) at 200 °C for 10 or 20 min reduced the levels of ochratoxin A by only 0-12% in the dried whole beans. Almost all of the ochratoxin A was infused into the coffee decoction when the roasted samples were ground and extracted with boiling water. Therefore, the reduction of ochratoxin A concentration of contaminated coffee beans by roasting under these conditions is ineffective. [ABSTRACT FROM AUTHOR]

Species of the Aspergillus ochraceus group were most abundant in the air of a coffee curing works of the seventeen environments studied at Mysore. They also occurred in large numbers in coffee dust, husk samples, sorghum and paddy. Incubating at room temperature (20-25°C) was found most suitable for enumeration of these species. Only 6.6% of the air-borne isolates of the group produced ochratoxin- A. A. ochraceus was the most common species of the group isolated from air and also yielded a greater proportion of toxic isolates. [ABSTRACT FROM AUTHOR]

The addition of NaBr to the fermentation of a marine isolate of the fungus Aspergillus ochraceus resulted in induced production of a new brominated mellein analogue. A new radical-scavenging halogenated isocoumarin, ( R)-(-)-5-bromomellein ( 1), and three known compounds, ( R)-(-)-mellein ( 2), clavatol ( 3), and circumdatin A ( 4), were isolated from the culture. The structure of 1 was assigned through a combination of spectroscopic data analyses. Compounds 1-4 exhibited mild radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH). The IC values of each compounds ( 1-4) were 24, 25, 30, and 32 μM, respectively, which were less active than the positive control, L-ascorbic acid (IC, 20.0 μM). [ABSTRACT FROM AUTHOR]