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The clinical and pathological features of Balkan (endemic) nephropathy are discussed and correlations of incidence with excess late summer and autumn rainfall outlined. Cultures of a strain of Penicillium verrucosum var. cyclopium isolated from maize collected in an endemic area were fed to rats and lesions were produced in the straight third segment of the proximal kidney tubules. Extensive degeneration and nuclear changes were seen and on prolonged feeding further nuclear enlargement (to greater than 6n) and the formation of multinucleate cells occurred. The relevance of these findings to the clinical disease in man, especially the occurrence of urinary tract tumours, and the evidence supporting mycotoxin involvement, are discussed.

Aflatoxin B1 (AFB1) is a carcinogenic metabolite produced by certain Aspergillus species. Ochratoxin A (OTA) is classified as “possible carcinogen” and it is a metabolite of Aspergillus ochraceus and Penicillium verrucosum. Fungi contaminate natural and processed olives which support AFB1 and OTA production. The aim of this study was to compare and investigate AFB1 and OTA production in three different varieties of damaged olives. For each variety two different treatments were applied: (1) olives with natural microflora and (2) olives inoculated with A. parasiticus after natural microflora elimination. AFB1 and OTA have been extracted simultaneously from olives, purified with immunoaffinity columns and quantitated by HPLC using fluorescence detector. The recoveries and detection limits of AFB1 and OTA were 94% and 0.15 ng AFB1 g−1 and 102.7%, 0.41 ng OTA g−1 respectively. Results showed that, meanwhile OTA was not found in any olive sample, AFB1 production within the three varieties of olives with natural microflora was significantly ( P ⩽ 0.05 ) different regarding their substrate and time of incubation (18 days). AFB1 production in two different varieties of black olives after inoculation by A. parasiticus was not significantly higher compared with control samples. On the contrary, AFB1 production in green olives was stimulated after the 12th day. Additionally, investigation on the occurrence of AFB1 and OTA in 30 samples of olives and olive pasta from Athens market showed OTA's presence in two samples of olives contaminated at the levels of 1.18 and 1.86 ng OTA g−1. Aflatoxin B1 was found at levels 0.15–1.13 ng AFB1 g−1 in all samples tested.

Ninety random grain samples were collected and analyzed for mycotoxins, and the effect of gamma irradiation on the production of mycotoxins in grains was studied. Aspergillus, Penicillium, Mucor, Rhizopus, Fusarium, Alternaria, Scopulariopsis and Cladosporium were the most common fungal genera isolated from grains. Aspergillus flavus, Aspergillus niger, Aspergillus candidus, Aspergillus ochraceus, Penicillium citrinum, Penicillium expansum, Penicillium citreonigrum, Penicillium purpurogenum, Penicillium griseofulvum and Penicillium verrucosum were the most common Aspergillus and Penicillium species in grains. Out of 120 Aspergillus and Penicillium isolates, 80 were mycotoxin producers. Analysis of grains revealed the occurrence of aflatoxin B 1 ochratoxin A, cycolopiazonic acid and citrinin. Of the 90 samples, 67 were positive for one or more mycotoxin. Irradiation of grains at dose of 2.0 and 4.0 kGy decreased significantly the total fungal counts compared with unirradiated controls. After 100 days of storage at room temperature, the unirradiated grains were contaminated with high concentrations of mycotoxins as compared with irradiated 4.0-kGy-samples. Mycotoxin production in grains decreased with increasing irradiation doses and was not detected at 6.0 kGy over 100 days of storage.

A Brazilian strain of Penicillium verrucosum was cultivated under different conditions in a two-step process, in order to verify the influence of nutrients, and of time periods of pre-fermentative and fermentative steps on the biosynthesis of metabolites. Extracellular and intracellular extracts were obtained from each culture in the four different production media used. Chemical profiles of the extracts were obtained by HPLC. Extract trypanocidal activities against trypomastigote forms of Trypanosoma cruzi were evaluated. The time period of incubation in the pre-fermentative and fermentative media, as well as the different nutrients tested, qualitatively and quantitatively modified the production of secondary metabolites by P. verrucosum, and the extract trypanocidal activities.

Ochratoxin A (OTA) is a secondary metabolite produced by several species of Aspergillus and Penicillium; among them Aspergillus ochraceus and Penicillium verrucosum are two ochratoxigenic species capable of growing in different climates and thus contamination of food crops with OTA can occur worldwide. OTA can be found in a wide range of foods such as cereals, coffee, cocoa, spices, beer, wine, dried vine fruit, grapes and meat products. OTA is toxic to animals, it presents neurotoxic, immunotoxic and nephrotoxic effects. It has been implicated in a human kidney disorder known as Balkan Endemic Nephropathy. This review focuses on the ecophysiology of ochratoxin-producing Aspergillus ochraceus and Penicillium verrucosum, the effect of environmental factors on their germination, mycelial growth, and OTA production. Knowledge of environmental conditions required for sucessive stages of fungal development represent the first step towards preventing mycotoxin formation. Predictive models for different stages of fungal development are presented, which allow prediction of the time before spoilage as a function of the abiotic factors. Finally, the implications of these studies in management of barley, coffee and grapes are described. This can help to identify the critical control points in their production, storage and distribution processes.

A PCR method for differentiation and detection of the two known ochratoxin A producing Penicillium species, Penicillium verrucosum and Penicillium nordicum has been developed. It is based upon two genes of the ochratoxin A biosynthetic pathway, namely the ochratoxin A polyketide synthase gene (otapksPN) and a non-ribosomal peptide syntethase gene (otanpsPN) from P. nordicum. Both ochratoxin A producing Penicillia differ characteristically in the PCR result, making a taxonomic differentiation possible. P. verrucosum gives consistently only a positive reaction with the primers for the otanpsPN gene, whereas P. nordicum is positive for both genes. The PCR reaction is negative with all of other food related fungal species tested. This PCR system has been used to analyse 62 Penicillium strains isolated from cured meat products or ripening rooms, the natural habitat of P. nordicum. Among the 62 analysed strains 11 (18%) were positive with all specific PCR reactions. All 11 strains were able to produce ochratoxin A. In a RAPD analysis performed in parallel all 11 strains showed a pattern characteristic of P. nordicum, indicating the congruence of all data. None of the other strains isolated from cured meat produced ochratoxin A; most of them (30 out of 62) had a RAPD pattern characteristic for Penicillium nalgiovense. Interestingly some of the P. nalgiovense strains showed weak PCR product bands with varying length after electrophoresis. This was true for both primer pairs. None of these P. nalgiovense strains however produced detectable amounts of ochratoxin A. A more detailed analysis revealed that P. nalgiovense carries similar but non-transcribed sequences to the ochratoxin A biosynthetic genes of P. nordicum.

The aim of this work was to study the combined effects of water activity (aw, 0.75-0.99), temperature (10-30 degrees C) and isolate on spore germination and mycelial growth of ochratoxigenic Penicillium verrucosum isolates on barley meal extract agar (BMEA). Lag phases prior to germination (h), germination rates (h(-1)) and growth rates (mm day(-1)) were obtained at each set of conditions and for all the isolates. Analysis of variance showed a significant influence of the abiotic factors assayed on fungal development. Minimum aw levels found for germination and mycelial growth were 0.80 and 0.85, respectively, while maximum germination and growth rates were obtained at 0.95-0.99 aw level. Although maximum germination was obtained at 20 degrees C, significant differences were not found between growth rates at 20 and 30 degrees C. Data were modelled by MLR regressions and response surface models were obtained. Spore germination and mycelial growth of ochratoxigenic P. verrucosum isolates are significantly affected by water activity and temperature. Moreover, different isolates may have slightly different response to environmental factors. Predictive models obtained may allow a rough prediction of germination/growth as a function of the storage temperature and moisture content of barley grains.

Biopreservation using polyphenols represents an alternative to chemical molecules for improving food safety. In this work, we evaluated the antifungal activity of polyphenols extracted from olive mill wastewater (OMWWP) to reduce or eliminate the growth of undesired fungi on the surface of dry fermented sausages. Antagonism against Penicillium expansum DSMZ 1282, Penicillium verrucosum DSMZ 12639, Penicillium nalgiovense MS01, Aspergillus ochraceus DSMZ 63304, Cladosporium cladosporioides MS12, and Eurotium amstelodami MS10 was evident at 1.25% OMWWP in vitro, whereas in situ application of 2.5% OMWWP strongly reduced undesired household fungal species such as C. cladosporioides, Penicillium aurantiogriseum, Penicillium commune, and Eurotium amstelodami, while a moderate antagonistic activity towards P. nalgiovense and Penicillium chrysogenum was observed at the same concentration. OMWWP at the concentrations used in this study demonstrated species-dependent antifungal activity by inhibiting both fungal growth and spore germination. Therefore, OMWWP can be regarded as a potential alternative to synthetic antifungal compounds to preserve the product from both oxidation and undesired fungi, without changing the sensory characteristics.

The natural mycobiota occurring in bee pollen is studied in the present report with special attention to analyze the incidence of fungal species that are potential producers of mycotoxins. A total of 90 ready-to-eat bee pollen samples were analyzed. Eighty-seven samples were collected in stores placed in different Spanish areas and three were from Buenos Aires (Argentina). The statistical results (ANOVA) showed that yeasts and Penicillium spp. were the predominant fungi. With regard to the potential mycotoxin producing species, Penicillium verrucosum, Aspergillus niger aggregate, Aspergillus carbonarius, Aspergillus ochraceus, Aspergillus flavus, Aspergillus parasiticus and Alternaria spp. were found. The last genus was isolated very frequently. The potential ability for producing ochratoxin A (OTA) and aflatoxins B(1), B(2), G(1) and G(2) was studied by culturing in vitro the isolates followed by analysis of these mycotoxins in culture extracts by HPLC with fluorescent detection. It was found that 100%, 53.3%, 33.3% and 25% of the isolates of A. carbonarius, A. ochraceus, P. verrucosum and A. niger aggregate, respectively, produced OTA. Moreover, 28.6% of the isolates from the A. flavus plus A. parasiticus group were able to produce aflatoxin B(1). Aflatoxin B(2) was detected in only 10% of the cultures. Aflatoxins G(1) and G(2) were not detected in cultures under the assayed conditions. This is the first report carried out on the natural mycobiota occurring in bee pollen in general and on the toxigenic capability of these isolates in particular.

Mycotoxin-producing fungi are natural contaminants of cereals and their toxins are harmful to humans and animals. Ochratoxin A (OTA) is among the most important. Heat treatment by drum drying does not eliminate already formed mycotoxins but the technique can reduce the number of viable fungi on the grain. The aim of this study was to establish a drying regime that kills fungal propagules on rye without reducing its quality for baking. Special attention was paid to some important mycotoxin-producing species. As drying temperatures and retention time in the drum are essential, the drum drier must be equipped with an effective control unit. Two different control systems were tested. The results showed that fungi colonising the grain can be much more efficiently and precisely reduced by controlling the maximum grain temperature (MGT) than the fixed constant drying air temperature (FAT). Drum drying very efficiently reduced the fungal propagules colonising the grain, including the mycotoxin-producing Penicillium verrucosum, Fusarium avenaceum, F. culmorum, F. poae, F. sporotrichioides and F. tricinctum. Both temperature and retention time in the drum affected survival rate of the fungi. Using MGT, a retention time of 10·5 min and a temperature of 64 °C killed 99% of the yeast propagules and 98% of the filamentous fungi. Moisture contents were reduced to about 12%. A similar drying regime in a supplementary trial reduced the number of P. verrucosum contaminated kernels from more than 70% to 12% but confirmed that drum drying did not destroy already formed OTA. The combination of a high drying capacity and a short but efficient heat treatment was obtained by drum drying as opposed to on-floor, batch and ordinary continuous flow drying, and it reduced the risk of mould deterioration to almost zero when the grain was properly stored afterwards. At the same time a high quality for baking was maintained. The highest baking quality in rye was obtained at grain temperatures of about 62 °C and only at grain temperatures above 70 °C visual quality changes were detected.

Ochratoxin A (OTA) is a secondary fungal metabolite produced by several moulds, mainly by Aspergillus ochraceus and by Penicillium verrucosum, that occurs in meat products. The aim of this work was to optimize an efficient extraction procedure for the determination of OTA in muscle tissue in order to assess its occurrence in meat samples. Three different apparatus, a Waring blender, a switching apparatus, and an ultrasonic processor, were evaluated to verify the efficiency of extraction. The analytical methods proposed involve the extraction with chloroform-orthophosphoric acid, cleanup through an immunoaffinity column, high-performance liquid chromatography/fluorescence detection for separation and identification of OTA, and confirmation with liquid chromatography/FD after methylation of OTA in muscle tissue. The limit of quantification of the proposed method was 0.04 microg kg(-1). Recoveries of OTA, using switching apparatus, ranged from 90.3 to 103.2% for chicken muscle spiked at 2.4 and 0.48 microg kg(-1), respectively, with a within-day relative standard deviation of 17 and 15.3%. The proposed method was applied to 38 chicken, swine, and turkey muscle samples and the presence of OTA was confirmed in five samples. Finally, the estimated daily intake of OTA in this study was between 23 pg kg(-1) body weight per day for swine samples and 18 pg kg(-1) body weight per day for turkey samples.

Rice samples collected from the Republic of Korea were analyzed for fungal mycoflora and mycotoxins: fumonisins, ochratoxin A, trichothecenes, and zearalenone. The potential of the fungi to produce each mycotoxin was also examined, so that the fungal isolates associated with mycotoxins occurring in rice could be verified. Penicillium citrinum and Aspergillus candidus were the most prevalent species infecting the samples, while Fusarium proliferatum was found as the dominant Fusarium species. Ochratoxin A was the most commonly detected mycotoxin analyzed in the present study; moreover, its level in some samples was above the EU tolerable limit (3 ng/g). According to rice culture experiments, it was revealed that in Korea, fumonisins detected in rice were due to F. proliferatum infection, whereas the occurrence of ochratoxin A was caused by Penicillium verrucosum, though there were no symptoms of disease in rice found in any sample. Furthermore, there appears to be an uneven geographical distribution of P. verrucosum as well as ochratoxin A in that most of them are found in the rice samples produced in the northern region of Korea.

Summary Penicillium nordicum is a fungal species able to produce high amounts of ochratoxin A. A 10 kb genomic DNA fragment of P. nordicum has been cloned which carries three long open reading frames. One open reading frame ( otapks PN) has homology to fungal polyketide synthases. The second open reading frame ( nps PN) has homology to non-ribosomal peptide synthetases and the third open reading frame ( asp PN) has homology to fungal alkaline serine proteinases. The non-ribosomal peptide synthetase and the polyketide synthase are convergently transcribed. Interestingly, the polyketide synthase can be identified by PCR only in P. nordicum strains and not in the related species Penicillium verrucosum or in ochratoxigenic Aspergillus species, indicating that the ochratoxin polyketide synthases are different in the important ochratoxigenic species. In contrast, the non-ribosomal peptide synthetase can be identified in P. nordicum and P. verrucosum , but not in other species. An inactivation of the polyketide synthase resulted in strains with abolished capacity to produce ochratoxin A. Expression of the polyketide synthase correlates with ochratoxin A biosynthesis.

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.

This study addresses the relationship between the ochratoxigenic strains of Penicillium verrucosum and ochratoxin A (OTA) contents in organically cultivated grain. It included 37 combined, non-dried grain samples from farmers with no drying facilities as well as 19 non-dried and 22 dried samples from six farms with on-farm drying facilities (Case studies 1-6). The study focused on the ancient wheat type spelt but also included samples of wheat, rye, barley, oats, triticale, emmer, and einkorn. All 78 samples were analysed for moisture content (MC) and occurrence of P. verrucosum. The latter was assessed by plating non-disinfected kernels on DYSG agar and counting those contaminated by the fungus. Fifty-five samples were analysed for OTA. Most of the combine harvested samples (82%) were contaminated with P. verrucosum prior to drying. This was ascribed to difficult harvest conditions and many samples of spelt, which was significantly more contaminated by P. verrucosum than oats, wheat and barley. Though not statistically significant, the results also indicated that spelt was more contaminated than rye, which is usually regarded the most sensitive small grain cereal. No correlation was found between number of kernels contaminated by P. verrucosum and OTA content. Despite many non-dried samples being contaminated by P. verrucosum, only two exceeded the EU maximum limit for grain (5 ng OTA g(-1)), both being spring spelt with 18 and 92 ng g(-1), respectively. The problems were most likely correlated to a late harvest and high MC of the grain. The case studies showed exceedings of the maximum limit in a batch of dried oats and spring wheat, respectively, probably to be explained by insufficient drying of late harvested grain with high MC. Furthermore, our results clearly indicate that OTA is not produced in significant amounts in samples with MCs below 17%. All dried samples with MCs above 18% exceeded the 5 ng OTA g(-1) limit in grain. However, no correlation between MC and the amount of OTA produced was found.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus and Penicillium verrucosum. It has been found and analysed in several foods and feeds. Owing to its toxicity and occurrence in food and feed, the European Community has issued directives and some countries have their own regulations for OTA contents in food, feed and beverages. This work describes a method for the determination of OTA in mulled and red wine. It is based on combined anion exchange/reversed-phase clean-up and was analysed by liquid chromatography coupled with tandem mass spectrometry (multiple reaction monitoring). The method was validated with natural contaminated and spiked wine samples with OTA contents from 1.34 to 3.48 microg kg(-1). Owing to its accuracy, good reproducibility and repeatability, this easy method is a good alternative to liquid chromatography-fluorescence detection methods.

Analytical methods are presented for detecting simultaneously 11 fungal metabolites (aflatoxins B1, B2, G1 and G2, citrinin, cyclopiazonic acid, mycophenolic acid, ochratoxin A, penicillic acid, penitrem A and roquefortine C) on different matrices. The methods were applied to determine the mycotoxins produced by different Penicillium crustosum, Penicillium nordicum and Penicillium verrucosum strains on yeast extract sucrose (YES) agar and cheese and bread analogues and are based on high-performance liquid chromatography (HPLC) and photodiode array detection (PDA). The growth substrate had a distinctive effect on the mycotoxin production ability of the fungi examined. The P. crustosum strains produced roquefortine C on all the substrates, with the highest amounts being detected on the cheese analogue. Penitrem A was synthesised on the cheese analogue only. The strains of P. verrucosum produced exclusively citrinin on YES, but both ochratoxin A and citrinin were detected in considerable amounts on the bread analogue. On the bread, toxin profiles varied significantly between the individual P. verrucosum strains. The cheese analogue was not favourable for the mycotoxin production of this species. The growth substrate had the least effect on the toxin production of the P. nordicum strains, which synthesised ochratoxin A in moderate amounts on all three media.

The ochratoxigenic mycobiota of grapes belonging to representative wine regions located along the Mediterranean coast of Spain at different developmental stages was identified. During the development of the berries, the occurrence of Aspergillus spp. increased while the percentage of berries contaminated by non-ochratoxin A (OTA) producing species such as Alternaria spp. and Cladosporium spp. decreased. Penicillium verrucosum, the only confirmed Penicillium spp. that is able to produce OTA, was not isolated. The contamination by OTA-producing species comes from the surface of the berries and not from the inner fruit. Black aspergilli were predominant among the different Aspergillus spp. isolated. All the Aspergillus carbonarius isolates were able to produce OTA at different concentrations. None of the isolates belonging to Aspergillus niger aggregate and to Aspergillus japonicus var. aculeatus were able to produce OTA. These results are a strong evidence of the contribution of A. carbonarius in the OTA contamination in wine grapes, mainly at the last developmental stages of the berries.

Studies were conducted to examine the effect of water activity (a(w), 0.97, 0.95, 0.93), pH (4.5, 6.0) and preservative type and concentration (potassium sorbate (KS), calcium propionate (CaP), 300 and 3000 ppm) on colonisation and ochratoxin A (OTA) production by three strains of Penicillium verrucosum on bread analogues. At 25 degrees C, colony extension from a point source was similar for all three strains at the different environmental treatments examined, with optimum growth at 0.97 and 0.96 a(w) and pH 6. Temporal OTA production over 36 days varied significantly with strain, pH and a(w) level. Maximum OTA was produced at approximately 28-36 days for two of the strains with an optimum at 0.93 a(w) at pH 6.0. One strain produced 75-80 microg g(-1) analogue under these conditions. However, at pH 4.5, optimum OTA was produced at 0.95 a(w) after a similar time period on bread analogues, with maximum amounts of about 25-35 microg g(-1) bread. KS and CaP incorporation inhibited growth completely at pH 4.5 and 3000 ppm at all three a(w) levels. However, at suboptimal concentration, little growth inhibition occurred. At pH 6, even with 3000 ppm, growth of all three strains occurred. Where effective inhibition of growth was achieved (3000 ppm/pH 4.5, all a(w) levels), no OTA was detected. However, when 300 ppm was used, up to 7 microg g(-1) was detected in bread analogues at pH 4.5. At pH 6, OTA was produced at all treatment conditions by all three strains, although preservative-treated bread had less present. Statistical analysis showed that many of the one-, two- and three-way interactions had a significant effect on growth and OTA production. These results suggest that there is a significant risk from OTA contamination if lower concentrations of existing preservatives are used in wheat-based bakery products contaminated by spoilage mycotoxigenic moulds such as P. verrucosum.

The major species of fungi responsible for ochratoxin production (OTA) in a range of commodities are Penicillium verrucosum, Aspergillus ochraceus and Aspergillus section Nigri, especially A. carbonarius. P. verrucosum is particularly important in northern Europe where damp cooler conditions occur and where drying regimes need to be efficient and effective for preventing post-harvest contamination with OTA. A. ochraceus can infect cereals including barley, maize, coffee, cocoa and edible nuts. A. carbonarius has been identified as the key species responsible for OTA contamination of grapes, wine and vine fruits. Recent studies have identified the environmental regimes, especially of temperature and humidity, which are conducive to growth and OTA production by these species pre- and post-harvest and during transport. The optimum and marginal interacting conditions for growth and OTA contamination often vary considerably. This has to be borne in mind when effective preventative management strategies are being implemented. Recent studies with cereals have suggested that isolation frequency can be related to OTA contamination of cereals. A logistical model has been developed and identified that 1000 CFUs g(-1) grain of P. verrucosum (CFUs) is the threshold limit at which the probability of exceeding the EU legislative limit of 5 microg kg(-1) in cereal grain can be predicted under different storage regimes. Controlled atmospheres >50% CO(2) is required to effectively prevent OTA accumulation in damp cereals. With regard to grapes, preharvest contamination with A. carbonarius has been shown to be a good indicator for regional risk in southern Europe from OTA contamination. The ecological conditions for optimum growth and OTA production have been shown to differ with the optima being 30-35 degrees C and 15-25 degrees C and 0.98-0.99 and 0.93-0.95 water activity, respectively. Studies on vine fruits (drying currants) suggest that OTA contamination and increase contamination levels occur during this drying process of 7-14 days. This will be influenced by prevailing weather conditions and drying rates. Minimizing OTA contamination in these and other commodities including coffee and cocoa require clear guidelines on safe moisture and temperature regimes pre- and post-harvest for the development of effective management strategies based on ecological criteria.

The influence of saprotrophic soil fungi: Aspergillus versicolor, Penicillium frequentans, Penicillium verrucosum var. cyclopium, and Trichocladium asperum on development, wholesomeness, gas exchange and yield of potato cv. Mila was studied in pot experiments during 1998–2000. The presence of each of the mentioned fungi species in soil accelerated potato germination and stimulated growth of overground plant parts in comparison with control plants. Additionally, the presence of the tested saprotrophes in soil prologed the potato growing period by inhibiting chloroses of necroses. The tested saprotrophic fungi also modified plant physiological processes, such as transpiration and assimilation. The contact of plant root system with soil saprotrophes diminished significantly assimilation and transpiration intensity of overground parts in comparison with the control plants, in all the years of the experiment. However, this response did not reduce the yield of tubers.

Sensors based on composites of metal oxides were fabricated and tested extensively under high-humidity and high-flow conditions with exposure to vapours reported to increase in the headspace of wheat grain (Triticum aestivum cv Hereward) colonized by fungi. The sensors that exhibited high sensitivity to target vapours combined with high stability were selected for inclusion into a four-sensor array prototype system. A sampling protocol aligned to parallel GC-MS (gas chromatography-mass spectrometry) and human olfactory assessment studies was established for use with the sensor system. The sensor system was utilized to assess irradiated wheat samples that had been conditioned to 25% moisture content and inoculated with pathogens known to cause spoilage of grain in storage. These included the fungi Penicillium aurantiogriseum, Penicillium vulpinum, Penicillium verrucosum, Fusarium culmorum, Aspergillus niger, and Aspergillus flavus and the actinomycete, Streptomyces griseus. The sensor system successfully tracked the progress of the infections from a very early stage and the results were compared with human olfactory assessment panels run concurrently. A series of dilution studies were undertaken using previously infected grain mixed with sound grain, to improve the sensitivity and maximize the differentiation of the sensor system. An optimum set of conditions including incubation temperature, incubation time, sampling time, and flow rate were ascertained utilizing this method. The sensor system differentiated samples of sound grain from samples of sound grain with 1% (w/w) fungus infected grain added. Following laboratory trials, the prototype sensor system was evaluated in a commercial wheat grain intake facility. Thresholds calculated from laboratory tests were used to differentiate between sound and infected samples (classified by intake laboratory technicians) collected routinely from trucks delivering grain for use in food manufacture. All samples identified as having an odour-related problem by the intake laboratory gave a total system output above the set threshold and were therefore rejected by the prototype system. A number of samples passed by the intake laboratory were rejected by the prototype system, resulting in what appeared to be false positive results. However, the thresholds were selected on the basis of a limited number of samples and may need to be adjusted to minimize false positives. The output from the sensor system was also compared with moisture content values for the wheat (where available) to demonstrate that the system was not simply measuring differences in moisture. A separate study (carried out at the intake facility) assessed 37 newly harvested wheat samples of different varieties and from different geographic locations within the UK. These samples were analysed by the sensor system, using the same thresholds as before. Six samples rejected by the system were then assessed by the wheat intake laboratory, where only one sample was rejected. This rejected sample had given the highest output when exposed to the sensor system. The commercial trial highlighted the promise of this prototype for the detection of spoilage in wheat grain and a larger trial should ascertain the reliability and long-term stability of the device and therefore confirm its usefulness to the industry.

Mycotoxin contamination of organically grown cereals has been a growing concern during recent years, one of the most important being ochratoxin A (OTA). In countries with a temperate, humid climate, OTA is produced by Penicillium verrucosum and it is crucial to focus on the fungus to obviate problems. This paper presents results to elucidate different aspects of the ecology of P. verrucosum. In a survey of differently farmed soils, P. verrucosum was found in 11 out of 65 soils (35% of the organically and 7% of the conventionally cultivated), these being the first reports of its natural occurrence in soil. Except for two soils it was found with low frequencies (100-300 cfu g dry soil-1), and the results point to home grown seed as a potential risk of soil contamination. In a survey of organically grown grain, P. verrucosum was found in 51% of the recently combined and not yet dried grain samples (60% of the rye and 53% of the wheat samples). This confirms that early contamination does take place and increases the demand for proper grain handling during drying and storage. Three case studies were performed at organic farms with different storage facilities. The results showed very clearly that there is no general risk of OTA contamination in organically cultivated grain. Within the same year, major differences between the farms were found and these differences were reproduced during three growing seasons. It therefore seems likely that an organic farming system, as such, does not present problems in relation to OTA contamination. It is, rather, certain management practices that are inappropriate and these management practices may be more prevalent in organic farming. This assumption is discussed in relation to home grown seed, crop rotation, organic fertilizers, exclusion of pesticides, and drying and storage facilities.

Fungi responsible for ochratoxin A (OTA) production have been studied especially on cereals, where Penicillium verrucosum and Aspergillus ochraceus are to be considered the main producers. Until 1998, these fungi were also believed to be responsible for the production of the toxin in grape, but OTA-producing A. carbonarius and A. niger were identified in dried vine fruits in 1999. Further studies pointed out that mycoflora potentially responsible for the presence of OTA in grapes are present in the field. Aspergilli are dominant to Penicillia, and among these Aspergilli section Nigri. A. carbonarius probably plays an important role because of the high percentage of positive strains and the amount of OTA produced. Aspergilli section Nigri are present on grape bunches early in the season and their frequency increases during later growth stages. At early veraison and ripening, the incidence of colonised berries is more related to the year than to the growth stage, but not to visible symptoms, since it is normal to isolate fungi from intact berries. Differences in ochratoxin content of berries have been detected between years, when the same vineyards, managed in the same way, showed high levels (1999) or the absence (2000) of the toxin. The results suggest that meteorological differences between years and grape-growing areas are responsible for differences in OTA levels, but the data are at present insufficient to draw firm conclusions.

Ochratoxin A (OA) is a mycotoxin detected in a variety of food and feeds mostly from countries with a temperate climate because of the fungi that produce it, mainly Aspergillus ochraceus and Penicillium verrucosum. In Argentina, there is no available information about the natural occurrence of OA and ochratoxigenic fungi from feedstuffs. The aim was to evaluate the natural occurrence of OA in poultry, pig and rabbit feeds over 8 months. Likewise, the capacity to produce OA by Aspergillus section Nigri was investigated. Mycotoxin analysis showed that in some months of sampling, OA was detected in three feeds. OA was found in 38% of the poultry feed samples tested with levels ranging from 25 to 30 ng g(-1). From rabbit feed samples, 25% contained OA and the levels ranged from 18.5 to 25 ng g(-1). Only 13% of the pig feed samples were contaminated with similar levels of toxins. Ninety-four black Aspergillus strains from feedstuffs were tested for OA production. Among these, the tested species were A. niger var. niger, A. niger var. awamori, A. japonicus var. japonicus, A. japonicus var. aculeatus and A. foetidus. For the detection of OA, three methodologies were applied: the two TLC methods used for the fast screening of the filamentous fungi for the production of OA were not sensitive enough to detect OA in any of the black Aspergillus strains. When an HPLC methodology was used, the results showed that 46% of the black Aspergillus strains were producers of OA, with levels ranging from 13 to 25 ng ml(-1) culture medium. The highest percentage of ochratoxicogenic strains was isolated from rabbit feeds with 100 and 78% of A. niger var. niger and A. niger var. awamori, with mean levels of 15.5 and 14.6 ng ml(-1), respectively. From pig feeds, 61% of the A. niger var. awamori were producers of this toxin with mean levels of 16 ng ml(-1). In poultry feeds, the lowest percentage of OA producer strains was detected. The results for the occurrence of OA in feeds from different sampling months depended on storage and humidity-temperature conditions. Therefore, a good storage practice becomes very important to prevent OA production

Mycobiota growing on food is often beneficial for the ripening and development of the specific flavor characteristics of the product, but it can also be harmful due to the production of undesirable compounds such as mycotoxins or antibiotics. Some of the fungi most frequently isolated from fermented and cured meat products such as Penicillium chrysogenum and Penicillium nalgiovense are known penicillin producers; the latter has been shown to be able to produce penicillin when growing on the surface of meat products and secrete it to the medium. The presence of penicillin in food must be avoided, since it can lead to allergic reactions and the arising of penicillin resistance in human-pathogenic bacteria. In this article we describe a study of the penicillin production ability among fungi of the genus Penicillium that are used as starters for cheese and meat products or that are frequently isolated from food products. Penicillium griseofulvum was found to be a new penicillin producer and to have a penicillin gene cluster similar to that of Penicillium chrysogenum . No other species among the studied fungi were found to produce penicillin or to possess the penicillin biosynthetic genes, except P. verrucosum , which contains the pcbAB gene (as shown by hybridization and PCR cloning of fragments of the gene) but lacks pcbC and penDE . Antibacterial activities due to the production of secondary metabolites other than penicillin were observed in some fungi.

The occurrence of P. verrucosum and ochratoxin A (OTA) were surveyed for 3 and 4 years, respectively. A total of 250 samples was collected from an average of 30 farms during the 2011, 2012, 2013 and 2014 winter seasons. Most storage bins surveyed were typically 11 m high round bins made of corrugated, galvanized steel, with flat-bottoms and conical roofs. Samples of clumped grain contained the most P. verrucosum (p

Mass spectrometric identification of proteins in species lacking validated sequence information is a major problem in veterinary science. In the present study, we used ochratoxin A producing Penicillium verrucosum to identify and quantitatively analyze proteins of an organism with yet no protein information available. The work presented here aimed to provide a comprehensive protein identification of P. verrucosum using shotgun proteomics. We were able to identify 3631 proteins in an "ab initio" translated database from DNA sequences of P. verrucosum. Additionally, a sequential window acquisition of all theoretical fragment-ion spectra analysis was done to find differentially regulated proteins at two different time points of the growth curve. We compared the proteins at the beginning (day 3) and at the end of the log phase (day 12).

Ochratoxin A (L-phenylalanine-N-[(5-chloro-3, 4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2 benzopyrane-7-yl)carbonyl]-(R)-isocoumarin) (OTA) one of the most studied mycotoxins is produced by Penicillium verrucosum and by Aspergillusochraceus but a few isolates of Aspergillus niger. It is one of the most important mycotoxin contaminants in agricultural products and continues to attract global attention concerning the hazard and impact on both human and animals due to its toxicity and occurrence. It has been detected in food commodities such as cereals, wine, coffee, figs, dried fruits and beer and in feeds for animals. The International Agency for Research on Cancer classifies it as a possible carcinogen to human (group 2B). Its symptom includes low appetite, weight loss, faintness, depression, high thirst and increased urination. OTA is one of the major causes of death in human beings and animals. These toxic fungi, contaminate food products in different phases of production and processing, especially in suitable environmental conditions viz., temperature and moisture conditions.

Despite the available data regarding antimicrobial activity of phenolic acids, studies dealing with the effects of their metabolites or derivatives are scarce. Therefore, the antimicrobial and demelanizing activities of Coprinopsis atramentaria extract, its organic acids, and methylated and glucuronated derivatives were evaluated. The antifungal activity was stronger than the antibacterial effects. In general, individual compounds (mostly organic acids) exhibited higher activity than the extract and even higher than the standards used in the assays. Methylated derivatives presented the highest demelanizing activity toward Aspergillus niger, A. fumigatus and Penicillium verrucosum var. cyclopium. The inclusion of methyl groups in the parental compound (CoAM1, CoAM2 and CoAM3) strongly increased antibacterial and antifungal activities of CoA, while the inclusion of acetyl groups (CoAGP) increased the antifungal activity but the antibacterial properties were maintained. For HA and CA, the inclusion of methyl groups (HAM1, HAM2, HAM3 and CAM) increased the demelanizing activity, but decreased the antimicrobial properties. The present work contributes to the knowledge of the mechanisms involved in the antimicrobial properties of organic acids namely, phenolic acids, usually present in mushrooms. Organic acids, and methylated and glucuronated derivatives could be used as antimicrobial agents., The authors are grateful to Fundacao para a Ciencia e a Tecnologia (FCT, Portugal) for financial support to the Portuguese NMR network and to FCT and FEDER-COMPETE/QREN/EU for the financial support through the research project PTDC/AGR-ALI/110062/2009 and the research centres (PEst-C/QUI/UI0686/2011 and PEst-OE/AGR/UI0690/2011). S. A. Heleno (BD/70304/2010) also thanks FCT, POPH-QREN and FSE for her grant. The authors also thank Serbian Ministry of Education, Science and Technological development (grant number 173032) for financial support.

Dry-cured sausages are colonised by moulds during the ripening process. The temperature and the salt content (which affects water activity, a w ) predispose the surface to colonisation by Penicillium species, including Penicillium nordicum and Penicillium verrucosum which can lead to contamination of the sausages with ochratoxin A (OTA). The objective of this work was to obtain scientific data on the impact that interaction between ionic water stress (a w ; 0.97, 0.94, 0.90, 0.87 and 0.84) and temperature (30, 25, 20, 15 and 10 °C) may have on lag phases prior to growth, growth and OTA production by some P. verrucosum and P. nordicum strains isolated from dry-cured meat products on a dry-cured sausage-based medium over a period of 12 days. Although P. nordicum had shorter lag phases than P. verrucosum , the latter grew faster than P. nordicum in most conditions tested. For both species, there was no growth and OTA production at 0.84 a w at all the temperatures tested. The fungi were more tolerant at moderate ionic a w conditions (0.94 and 0.90) and 20 and 25 °C. In contrast, the patterns of production of OTA were very different from those for growth. Different OTA production profiles between the two OTA-producing species were found. While P. nordicum began producing OTA in most of the conditions tested by day 6, P. verrucosum only produced the toxin in these conditions when the temperature and a w were > 10 °C and > 0.90, respectively. However, the P. verrucosum strain produced much higher concentrations of OTA than the P. nordicum strain in all conditions. We developed contour maps of the optimum and marginal a w × temperature conditions for growth/OTA production on dry-cured sausage-based medium for the first time. This suggests that these interacting conditions during the early phases of production must be effectively controlled as these favour growth of the toxigenic Penicillia . Knowledge on the ecophysiology of these two important Penicillium species on these matrices could help to make appropriate technological modifications during the sausage ripening process. Thus, our findings may help in informed decision-making in relation to temperature and salt additions at the beginning of processing/curing. Such changes may favour colonisation of starter cultures over OTA producing Penicillia and minimise OTA contamination risks in dry-cured sausages. This may be then effectively incorporated into the hygienic production system in the framework of HACCP.

Biocontrol using autochthonous Debaryomyces hansenii isolates is a potentially suitable strategy for inhibiting toxigenic moulds in dry-cured meat products. The antifungal activity of 280 D. hansenii isolated from dry-cured meat products as well as the mode of action of the most active isolates against toxigenic penicillia were evaluated in this work. A 13.9% of the D. hansenii isolates showed inhibitory activity in a radial inhibition assay. The effects on penicillia growth of both the cell-free culture filtrate and volatile compounds from active yeast isolates were analysed. Penicillia growth inhibition by D. hansenii was probably based on additive or synergistic effects of several inhibiting factors such as competition for nutrient and space, and production of soluble or volatile compounds. When four D. hansenii isolates were tested on dry-fermented sausage, two of them produced a significantly growth reduction of the ochratoxigenic Penicillium verrucosum , keeping its counts under the level considered as hazardous for the mycotoxin presence. Therefore, the use of these two D. hansenii isolates during the processing of dry-fermented meat product could be a promising tool to control toxigenic moulds in the meat industry.

Mycotoxins are secondary metabolites produced by several species of fungi during colonization and growth on plants. The occurrence of mycotoxins in commodities represents a major threat to humans and animals. Their formation in the infected crops and persistence in food and feed is associated with different toxicities including mutagenicity and estrogenic, gastrointestinal, urogenital, vascular, kidney and nervous disorders as well as the induction of cancer. Some mycotoxins are also immuno-compromising, and can thus reduce resistance to infectious agents (Desjardins, 2006). Moreover, significant economic losses are associated with the impact of mycotoxins on human health, animal productivity, and both domestic and international trade. It is estimated that 25% of the world food production, including many basic foods, are affected by mycotoxins producing fungi, while cereals appear to be contaminated to even higher levels (CAST, 2003). In particular, wheat is a crop that can be commonly contaminated by three of the most important mycotoxin-producing fungi: Aspergillus ochraceus, Penicillium verrucosum and Fusarium graminearum (Marasas et al., 2008). Therefore, three of the most important mycotoxins occurring in agricultural products, ochratoxin (produced on wheat mainly by A. ochraceus and P. verrucosum) and deoxynivalenol (DON) and zearalenone (ZEA), both produced on wheat mainly by F. graminearum, can be common contaminants of wheat (Miller et al., 1995). The detection and control of each toxigenic fungal species in planta is key to reduce fungal contamination and in turn prevent mycotoxins from entering the human food and animal feed.

Eterična olja origana (Origanum vulgare L.), mete (Mentha piperita L.), janeža (Foeniculum vulgare Mill.), in borovih (Abies alba Mill.) iglic in storžev, ter njihove sestavine timol, karvakrol, mentol in anisaldehid smo testirali kot snovi z protiglivno aktivnostjo proti plesnim vrste Penicillium verrucosum. Najnižje minimalne inhibitorne koncentracije (MICs) smo določili eteričnemu olju origana, ter karvakrolu, timolu, in mentolu. Rast plesni vrste P. verrucosum je bila popolnoma inhibirana po 21 dneh inkubacije v prisotnosti ½ MIC timola (0,0625 mg mL-1), medtem ko so eterično olje origana (½ MIC, ¼ MIC), timol (¼ MIC) in karvakrol (½ MIC, ¼ MIC) inhibirali rast plesni od 8,3 % do 70.8 %. Dodatek mentola v koncentracijah ¼ MIC in ½ MIC (0.1875 mg mL-1 in 0.3750 mg mL-1) pa ni imel inhibitornega vpliva na rast plesni vrste P. verrucosum. Popolno inhibicijo tvorbe ohratoksina A smo določili le v primeru, ko je bila tudi rast teh plesni popolnoma inhibirana (pri ½ MIC timola). Največjo inhibicijo tvorbe ohratoksina A (96,9 %) po 21 dnevih inkubacije smo določili pri ¼ MIC timola (0.0313 mg mL-1). Pospešeno tvorbo ohratoksina A (od 13,9 % do 28,8 %) pa smo določili pri ¼ MIC eteričnega olja origana (0.2930 mg mL-1) in ½ MIC karvakrola (0.1953 mg mL-1). Protiglivni vplivi so bili odvisni od testirane snovi, njene koncentracije ter časa interakcij med protiglivno snovjo in plesnimi vrste P. verrucosum. Rezultati nakazujejo možnosti uporabe eteričnega olja origana v živilih namesto kemijskih konzervansov. Ker pa so živila kompleksna okolja v katerih je potrebno upoštevati več dejavnikov, so potrebne nadaljnje raziskave., Essential oils from oregano (Origanum vulgare L.), mint (Mentha piperita L.), fennel (Foeniculum vulgare Mill.), and pine (Abies alba Mill.) needles and cones, and their active substances thymol, carvacrol, menthol, and anisaldehyde were tested for antifungal activity against Penicillium verrucosum. The lowest minimal inhibitory concentrations (MICs) were achieved for essential oil of oregano, followed by carvacrol, thymol, and menthol. These antifungal components were further investigated, as the main aim of our study was to elucidate the effect of natural antifungals on ochratoxin A production. During 21 days of exposure, the growth of P. verrucosum, and subsequently the production of ochratoxin A, was fully inhibited by thymol at ½ MIC (0.0625 mg mL-1), but menthol at ¼ and ½ MIC (0.1875 and 3750 mg mL-1) showed no growth inhibition. After 21 days of incubation, the greatest inhibitory effect on ochratoxin production (inhibition was 96.9 %) was also achieved with thymol at ¼ MIC (0.0313 mg mL-1). Essential oil of oregano (¼ MIC, 0.2930 μL mL-1) and carvacrol (½ MIC, 0.1953 μL mL-1) stimulate production of ochratoxin A at 13.9 % to 28.8 %, respectively. The observed antifungal effects depended on the agent, the concentration used, and the time of interaction between the agent and P. verrucosum. Our results indicate the possibility of using oregano essential oil as a substitute for artificial preservatives in certain foods, but further research is needed.

Ochratoxin A (OTA) is produced by Aspergillus westerdijkiae , Aspergillus carbonarius , and Penicillium verrucosum , or species closely related to each of those. It is known to affect many animal species and is a powerful kidney toxin. However, its effects, if any, on humans at current levels of exposure remain unclear. It is regarded by the International Agency for Research on Cancer as possibly carcinogenic to humans. OTA was once considered to cause the disease known as Balkan endemic nephropathy, but that now seems unlikely. It is most commonly found in European and Canadian cereals, but also occurs in coffee, cocoa, dried vine fruits and wines. Levels in the diet rarely approach the provisional tolerable weekly intake level established by the Joint FAO/WHO Expert Committee on Food Additives.

The relationship between viable mould count, ergosterol content and ochratoxin A (OA) formation was studied at different inoculum concentrations of Aspergillus ochraceus NRRL 3174 and Penicillium verrucosum NRRL 3260 grown on sterile long-grain enriched white rice as the substrate. Ergosterol was determined by extraction, saponification and quantification using high performance thin layer chromatography (HPTLC) with UV detection. Ergosterol and ochratoxin A were detected after 3 days of incubation and reached their maximum at 7-10 days of incubation. After that, a decline in the concentrations in both ergosterol and ochratoxin was observed. Ergosterol measurement by HPTLC appeared to be a useful method to detect fungal activity, which corresponded to ochratoxin production. Thus, the ergosterol assay may have a use as an early indicator of potential mycotoxin production.

A respiratory syndrome very similar to extrinsic allergic alveolitis due to Penicillium verrucosum was recognized in 4 workers employed in a Gorgonzola cheese factory. A mycogen allergy to P. verrucosum, used as starter in the production, was demonstrated by positive sputum culture and detection of specific antibodies in the blood. Intense and prolonged exposure to inhalation of fungal spores could have lead to the development of this allergic response. The fact that 2 of the subjects are siblings seems to indicate host susceptibility or immunological constitution in the pathogenesis of the respiratory allergy.

Farm workers are often exposed to high concentrations of airborne organic dust and fungal conidia, especially when working with plant materials. The purpose of this investigation was to study the possibility of exposure to the mycotoxin ochratoxin A (OTA) through inhalation of organic dust and conidia. Dust and aerosol samples were collected from three local cowsheds. Aerosol samples for determination of total conidia and dust concentrations were collected by stationary sampling on polycarbonate filters. Total dust was analysed by gravimetry, and conidia were counted using scanning electron microscopy. A method was developed for extraction and determination of OTA in small samples of settled dust. OTA was extracted with a mixture of methanol, chloroform, HCI, and water, purified on immunoaffinity column, and analysed by ion-pair HPLC with fluorescence detection. Recovery of OTA from spiked dust samples (0.9–1.0 μg/kg) was 74% (quantitation limit 0.150 μg/kg). OTA was found in 6 out of 14 settled dust samples (0.2–70 μg/kg). The total concentration of airborne conidia ranged from < 1.1 × 104 to 3.9 × 155 per m3, and the airborne dust concentration ranged from 0.08 to 0.21 mg/m3. Conidia collected from cultures of Penicillium verrucosum and Aspergillus ochraceus contained 0.4–0.7 and 0.02–0.06 pg OTA per conidium, respectively. Testing of conidial extracts from these fungi in a Bacillus subtilis bioassay indicated the presence of toxic compounds in addition to OTA. The results show that airborne dust and fungal conidia can be sources of OTA. Peak exposures to airborne OTA may be significant, e.g., in agricultural environments.

The aim of the study was to develop a new screening method to detect growth and ochratoxin A (OTA) production by multiple fungi growing in a small quantity of culture media, using microtiter plates. Eight ochratoxigenic species were included in the study. The strains were inoculated in sterile 96-well flat-bottom microtiter plates containing Yeast Extract Sucrose broth and Czapek Yeast Extract broth and incubated at 25 °C. Growth was daily monitored by absorbance measurements for 4 days and extended to 7 and 10 days for Penicillium spp. The entire experiment was repeated twice on different days. On each sampling time, five of the seven replicate wells inoculated for each strain and culture media were randomly selected and the content of each well was removed, extracted and injected into the HPLC. No statistically significant differences were observed for absorbance and OTA values, neither between replicates nor between experiments. Quantifiable OTA levels were detected after 48 h of incubation in Aspergillus alliaceus , Aspergillus carbonarius and Aspergillus niger , after 72 h in Aspergillus flocculosus , Aspergillus steynii and Aspergillus westerdijkiae and after 7 days in Penicillium nordicum and Penicillium verrucosum . The method offers the necessary tools for a rapid detection of growth and OTA production avoiding the use of plate cultures and can be very useful when many fungal isolates need to be screened.

Cereals are among the most vulnerable substrates to mycotoxin contamination. One of the major fungal species colonising the ecosystem of stored grain is Penicillium verrucosum because of its ability to produce ochratoxin A (OTA). Knowledge of favourable environmental conditions promoting fungal growth and mycotoxin biosynthesis seems to be a crucial step towards preventing mycotoxin formation in stored grain. The aim of the study was to examine the effect of temperature and different cereal substrates (wheat, triticale, rye, barley, maize, rice) on fungal growth and OTA and citrinin (CIT) production by a mycotoxin-producing P. verrucosum strain at one moisture content. The correlation between fungal growth and mycotoxin biosynthesis was also investigated. For this purpose, cereal substrates moistened, autoclaved and inoculated with P. verrucosum spores were stored at 10°C, 20°C and 30°C for 40 days. After the storage period, the ergosterol (ERG) content as a fungal growth biomarker and OTA and CIT levels were determined using HPLC. Fungal growth was observed on all cereals at each temperature. Maximum ERG levels were found at 30°C and the values ranged from 401 to 3280 mg kg(-1) of grain. Among tested cereals, the highest ergosterol content was found on rice at all applied temperatures. Accumulation of OTA and CIT was detected in samples stored at 20°C and 30°C, while optimal conditions for the production of both mycotoxins were observed at 20°C. The maximum concentration of OTA (31.9 ± 4.3 (SD) μg kg(-1)) and CIT (74.8 ± 5.7 μg kg(-1)) were found on rice. The ability to biosynthesise OTA and CIT at 10°C was not detected. Analysis of variance showed that single factors, temperature and cereal matrices and their interactions had significant effects on ERG, OTA and CIT levels. Optimal conditions for fungus growth did not coincide with the ones for mycotoxin formation.

Speck is a ham specialty product traditionally produced in South Tyrol (Italy) and North Tyrol (Austria) by farmers, butcheries, and meat industries. To date, nothing has been learned about fungi associated with this smoked and cured meat. Therefore, it was the main objective of this study to assess the typical mycobiota of Speck in relation to the different production types and the geographic provenance. A total of 121 Speck samples from North Tyrol and South Tyrol was analyzed. From 63 isolated fungal species, only a few can be regarded as typical colonizers: Eurotium rubrum and Penicillium solitum were the dominating species in all types and parts of Speck (crust, meat, and fat). Eight other Penicillium spp. were relatively frequent. The species diversity increased from industrially produced Speck to products from butcheries and farmers, and it was higher in all types of South Tyrolean products. Among the typical mycobiota, Penicillium verrucosum, Penicillium canescens, and Penicillium commune are known as potentially mycotoxigenic.

Cryptoendolithic microbial communities, living in porous sandstone rocks in the McMurdo Dry Valleys (Ross Desert) of Southern Victoria Land, Antarctica, were found within weathered pegmatite rocks in Northern Victoria Land, and the first endemic Antarctic fungal genus Friedmanniomyces endolithicus anam.‐gen. and sp. nov. was isolated from this community. Selected microfungi from these communities and from soil were examined for the production of extracellular enzymes and antibiotic substances. The cryptoendolithic strain CCFEE 5001 was particularly remarkable for consistent glycosidase activity, coupled with barely detectable growth. Chitinase activity was highest in the soil mi‐crofungus Verticillium cfr. lecanii (CCFEE 5003). This strain and its purified chitinase were active on Mucor plumbeus, Cladospori‐um cladosporioides, Aspergillus versicolor, and Penicillium verrucosum, producing mycelial damage and cell lysis. The strain CCFEE 5020, isolated from rocks, showed antibiotic activity against...

Growth of commonly occurring filamentous fungi in foods may result in production of mycotoxins, which can cause a variety of ill effects in humans, from allergic responses to immunosuppression and cancer. According to experts, five kinds of mycotoxins are important in human health around the world: aflatoxins, ochratoxin A, fumonisins, certain trichothecenes, and zearalenone. These toxins are produced by only a few species of fungi, in a limited range of commodities. Aflatoxins are potent carcinogens, produced by Aspergillus flavus and A. parasiticus in peanuts, maize and some other nuts and oilseeds. Ochratoxin A is a kidney toxin and probable carcinogen. It is produced by Penicillium verrucosum in cereal grains in cold climates, by A. carbonarius in grapes, wines and vine fruits, and by A. ochraceus sometimes in coffee beans. Fumonisins, which may cause oesophageal cancer, are formed by Fusarium moniliforme and F. proliferatum, but only in maize. Trichothecenes are highly immunosuppressive and zearalenone causes oestrogenic effects; both are produced by F. graminearum and related species. Current reporting probably underestimates the effect of mycotoxins as a cause of human mortality.

A field experiment was conducted to elucidate the survival of P. verrucosum in infested bulk soil (T1) and infested soil with waste grain (T2). The infested soil and reference soil (T3) was filled into steel cylinders, which were buried and sampled 13 times during a period from October 1994 to March 1996. The abundance of P. verrucosum and indigenous soil fungi were assessed by dilution plating on a selective and diagnostic medium (DYSG). Kernel infection was examined in T2. According to our results, P. verrucosum seems well adapted to survival in arable soil and little affected by indigenous fungi. During the first autumn and winter the grain caused a proliferation of P. verrucosum while its abundance in bulk soil was more constant except for a decrease in February 95, which is ascribed to frost/thaw alternations. In T2, P. verrucosum initially infected more than 50% of the kernels but during the first few months it was ousted by other fungi. A hypothesis regarding waste grain as the natural niche for the fungus in the field was therefore partly rejected. A gradual decrease in the abundance of P. verrucosum in soil during spring, a die-off in the dry summer and a proliferation during the second winter were found in both T1 and T2. Our results cannot provide the reason for the increase during the second winter. On an overall basis, however, they show that P. verrucosum can survive in the field, proliferate on soil organic matter and probably become an integral part of the soil ecosystem. This may constitute a risk of grain contamination when given appropriate environmental conditions.

Growth and ochratoxin A accumulation by two isolates of Penicillium verrucosum, IBT5010 and IBT12803, were examined in co-cultures with the antagonistic yeast Pichia anomala or baker's yeast Saccharomyces cerevisiae. Each yeast was co-cultured with Peniciilium verrucosum in malt extract agar supplemented with 1% yeast extract and incubated at 25 °C for 14 d. Ochratoxin A was found to be stable at 25°, whether or not yeast was present. Pichia anomala and Saccharomyces cerevisiae reduced mould growth to about the same extent in vitro. Both yeasts significantly reduced the growth of Penicillium verrucosum isolates at 3 x 103 yeast c.f.u./plate. In all co-cultures in vitro accumulated ochratoxin A was reduced to below the detection limit (100 ng/plate) by yeast amounts as low as 3 x 102 c.f.u./plate. In wheat, the growth of Penicillium verrucosum IBT5010, measured as c.f.u., was not reduced in the presence of Pichia anomala, whereas the growth of Penicillium verrucosum IBT12803 was clearly inhibited by this yeast. A similar pattern was observed for ochratoxin A accumulation in wheat when the two mould strains were co-cultured with Pichia anomala. Mycotoxin production was more sensitive to the presence of yeasts than was mould growth. Co-cultivation with yeasts gave no detectable stimulation of ochratoxin A accumulation in agar or wheat.

The natural occurrence of ochratoxin A in grain samples of 23 rice cultivars was in the range 0.01-1.0 ng g(-1) rice. Samples from the same cultivars were surface sterilized with NaClO, dried to 19% water content and equilibrated at water activity (a(w)) 0.75 and 20 degrees C for 8 days. Varietal differences in equilibrium w/w water content (p < 0.0001) were found, reflected by differences in amylose and protein contents. Samples were then inoculated with an isolate of Penicillium verrucosum with 1 ml spore suspension to each 50 g rice sample; and incubated at a(w) 0.75 and 20 degrees C for 23 weeks. During incubation, ochratoxin A was accumulated in all cultivars. Significant varietal differences in ochratoxin A accumulation were observed (p < 0.0001). Grain samples with less than 19.5% equilibrium water content accumulated less ochratoxin A (p < 0.005). In a multiple regression analysis accumulated ochratoxin A content was expressed as a function of natural occurrence of ochratoxin A (p < 0.05), equilibrium water content at time of inoculation (p < 0.005), 1000-grain weight (p < 0.1), and chalkiness of endosperm (p < 0.05), with p < 0.0001 for the full function. Naturally occurring ochratoxin A was the strongest independent variable with p < 0.0005 for the slope coefficient in single regression. Rice cultivars IR8, IR24, IR620030-18-2-2 and R91-1081-1 had exceptionally low accumulation of ochratoxin A.

Macroscopic nephropathy was observed in 506 pigs at slaughter in Bulgaria in 1993/94. Histopathological changes were mainly degenerative and proliferative, and were linked with kidney hypertrophy similar to that of the classical Danish Syndrome. Retention cysts formed by dilated tubules, activation or proliferation of capillary and vascular endothelium, and the development of neoplastic tissue were also observed. The most advanced pathology took the form of extensive interstitial fibrosis. Traces of ochratoxin A were found in the kidneys of the majority of 96 cases examined, and in some feed samples taken retrospectively from farms or commercial sources. The dietary ochratoxin concentration (100 micrograms/kg), calculated from serum analyses, closely matched the average of individually analysed feeds. In other feeds no ochratoxin A was detected and the cosmopolitan mycobiota isolated did not include the ochratoxinogenic Penicillium verrucosum that caused the Danish syndrome. Aspergillus ochraceus was rare and the isolates did not synthesise ochratoxin in laboratory culture. The unconfirmed diagnosis of ochratoxicosis suggests a complex or multi-toxin aetiology for this rather common chronic disease in Bulgaria.