Penicillium Verrucosum Research Articles

Monitoring of Ochratoxin A biosynthesis related genes in Penicillium verrucosum by differential display reverse transcription PCR (DDRT-PCR) experiments.

Differential display reverse transcription polymerase chain reaction experiments were performed to monitore Ochratoxin A biosynthetic genes inPenicillium verrucosum BFE487. A stringent prerequisite for the performance of this type of experiments is the knowledge of the differential expression of OTA genes. Differentially expressed total RNA from permissive/restrictive growth conditions and alternatively OTA(+) wild type/OTA mutant strains have been isolated, reverse transcribed and used as a template in DDRT-PCR experiments. About 70 differentially expressed cDNA fragments could be isolated, cloned and sequenced.

MYCOMON-monitoring project of toxigenic fungi in food in years 1999 – 2001

An occurrence of the toxigenic fungi producing aflatoxins and ochratoxin A in food was investigated in the study ("MYCOMON") in years 1999-2001. Twenty five commodities were purchased from retail in twelve collection places in the Czech Republic (300 food samples together). The presence of potentially toxigenic fungiAspergillus flavus was observed in 28% of the sampled food (black pepper, black tea, caraway seeds, fine flour, fruit tea, oat flakes) in the year 1999, in 17% of the sampled food (black pepper, black tea, caraway seeds, fine flour, fruit tea, oat flakes) in the year 2000 and in 18% of the sampled food (black pepper, black tea, caraway seeds, fine flour, fruit tea, oat flakes) in the year 2001.Aspergillus tamarii (aflatoxins producer) was found in black pepper samples (25%) in year 1999, in black pepper samples and black tea (25%) in year 2000 and in black pepper samples and black tea (21%) in year 2001.Aspergillus parasiticus andAspergillus nomius were not isolated.Aspergillus sectionNigri (potential producer of ochratoxin A) was detected in some food (black pepper, black tea, caraway seeds, fine flour, fruit tea, raisins, sweet red pepper).Penicillium verrucosum and Aspergillus ochraceus were not isolated from the tested food.

Production of penicillin by fungi growing on food products: identification of a complete penicillin gene cluster in Penicillium griseofulvum and a truncated cluster in Penicillium verrucosum.

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.

Methods of analysis for ochratoxin A.

The mycotoxin ochratoxin A (OTA) is produced by the fungi Aspergillus alutaceus and Penicillium verrucosum and has carcinogenic, nephrotoxic, teratogenic and immunosuppressive properties. The levels of OTA in foodstuffs are regulated in several countries, so reliable and sensitive methods are necessary for its determination. Procedures for extraction of OTA from ground foods generally use an organic solvent in the presence of acid or an extraction solvent containing aqueous sodium bicarbonate. Cleanup procedures include partition into aqueous sodium bicarbonate, solid phase extraction (SPE) columns and immunoaffinity chromatography. The latter technique allows detection of sub-ppb levels of OTA in a wide variety of foods and in plasma. The most widely used determinative procedure is reversed phase liquid chromatography (LC) with detection by fluorescence (excitation 330-340 nm, emission 460-470 nm) or, more recently, by tandem mass spectrometry. ELISA methods are also available. Certified reference materials containing OTA have been prepared.

Relationship of mould count, ergosterol and ochratoxin A production

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.

Behavior of Ochratoxin A in Brewing

Malt containing high levels (up to 52 μg/kg) of ochratoxin A (OA) was prepared on a pilot scale by inoculating malting barley with live Penicillium verrucosum. This malt was then used for pilot brewing trials to determine the extent to which OA can survive the brewing process. Substantial losses, up to 40% of the OA in the grist, were observed during mashing, most probably by proteolytic degradation. Another 16% was eliminated with the spent grains. Some losses of OA occurred during fermentation, but these were less than those in mashing. Overall, between 13 and 32% of the OA present in the original grist survived into the beer.

Ochratoxin A in airborne dust and fungal conidia.

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, HCl, 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 microg/kg) was 74% (quantitation limit 0.150 microg/kg). OTA was found in 6 out of 14 settled dust samples (0.2-70 microg/kg). The total concentration of airborne conidia ranged from < 1.1 x 10(4) to 3.9 x 15(5) 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 Mycobiota of Speck, a Traditional Tyrolean Smoked and Cured Ham

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.

Ecology and biology of microfungi from Antarctic rocks and soils

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 Pseudomonas putida, Sarcina sp., Bacillus subtilis, and Escherichia coli, under the test conditions. Good antibiotic activity was produced in the temperature range of 5° to 25° C, pH 4.0, agitation at 700 rpm, and areation at 1.0 vvm, in media containing 0.5% substrate sugar, though this supported only minimal growth. The investigation of soil microfungi in contrasting sites at the BIOTAS area at Edmonson Point indicated that both the presence of bird colonies and changes in temperature and UV exposure obtained by using plastic cloches influenced species composition and density. A ‘Culture Collection of Fungi from Extreme Environments’ (CCFEE) has been established at the University of Tuscia, housing fungal strains from Antarctica.

Toxigenic fungi: which are important?

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.

UV-Guided isolation of verrucines A and B, novel quinazolines from penicillium verrucosum structurally related to anacine from penicilliumaurantiogriseum

Two novel quinazolines, verrucines A (1) and B (2), have been isolated as a major and a minor metabolite, respectively, of Penicillium verrucosum. Both are condensates of one mole each of anthranilic acid, phenylalanine, and glutamine. The structures were elucidated by spectroscopic methods, and the two compounds were found to be epimers. The spectroscopic data for the ostensible benzodiazepine anacine reported from Penicillium aurantiogriseum, composed of one mole each of anthranilic acid, leucine, and glutamine, appeared very similar to those of 1. Therefore, a revised quinazoline structure for anacine is proposed.

Detection and estimation of conidial abundance of Penicillium verrucosum in soil by dilution plating on a selective and diagnostic agar medium (DYSG)

Penicillium verrucosum is one of the main producers of ochratoxin A (OA) in agricultural commodities. To forecast the risk of OA contamination, there is a need to improve our knowledge on the ecology of P. verrucosum in the field. Dilution plating on ‘dichloran yeast extract sucrose 18% glycerol agar’ (DYSG) offers a simple and very sensitive method of detecting P. verrucosum propagules in soil. The properties of DYSG are illustrated in a suspension mixture experiment and confirmed in a soil mixture experiment. In the latter, P. verrucosum could be detected in conidial concentrations below 200 colony forming units (cfu) g−1 soil even when it constituted no more than 0.3% of the cfu. Furthermore, the DYSG method can be used to estimate the abundance of P. verrucosum propagules in soil with good precision. In some of the analysed cases, however, it was necessary to use appropriate mathematical models to treat results with high numbers of cfu on the Petri dishes.

Ochratoxin A accumulation in cultures of Penicillium verrucosum with the antagonistic yeast Pichia anomala and Saccharomyces cerevisiae

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.

Chitinolytic activity at low temperature of an Antarctic strain (A3) of Verticillium lecanii

The chitinolytic activity of Verticilkium cfr. lecanii A3, a strain isolated from continental Antarctica, was compared to those of two selected strains of Trichoderma harzianum. After 72 h of incubation at 25°C in media containing chitin as the sole carbon source, all strains showed the same enzyme activity (ca. 230 mU/ml); at 15°C, the levels of enzyme activity of the three strains were similar to those obtained at 25°C. At 5°C, in contrast, the activity of V. lecanii was ca. 4 times higher than those of both strains of T. harzianum (203 and 57 mU/ml, respectively; incubation time 144 h). The chitinase of V. lecanii, purified by preparative isoelectric focusing and ion-exchange chromatography, was shown to be a glycoprotein with apparent molecular weight of 45 kDa and isoelectric point of 4.9. The enzyme was active over a broad range of temperatures (5–60°C): at 5°C, its relative activity was still 50% of that recorded at 40°C (optimal temperature). V. lecanii and its purified chitinase showed clear inhibitory effects on the growth of some test moulds such as Mucor plumbeus, Cladosporium cladosporioides, Aspergillus versicolor and Penicillium verrucosum: observations under the light and scanning electron microscopes revealed that growth inhibition was accompanied by mycelial damage and cell lysis.

Ochratoxin A in rice cultivars after inoculation of Penicillium verrucosum.

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.

Ochratoxin a in rice cultivars after inoculation of Penicillium verrucosum

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 (aw) 0.75 and 20 °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 aw 0.75 and 20 °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. Copyright © 1998 John Wiley & Sons, Ltd.

Porcine nephropathy in Bulgaria: a progressive syndrome of complex or uncertain (mycotoxin) aetiology

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...

Does the mycotoxin citrinin function as a sun protectant in conidia from Penicillium verrucosum.

Our results demonstrate high concentrations of the UV absorbing mycotoxin citrinin in the outer layer of spores from three citrinin-producing strains of Penicillium verrucosum, which is released in an aqueous environment. An important function of the toxin could be to act as a sun protectant in order to create favorable conditions during the initial germination process. When spores from these strains of P. verrucosum were examined by confocal microscopy, a clearly visible fluorescent layer associated with the cell wall was observed. The strains were grown on agar plates, and the mycelial mat was washed with saline. This suspension contained at least 95% of the spores and particulate material, which was removed by filtration after counting the conidia. An aliquot of this filtrate was extracted and citrinin was purified by high pressure liquid chromatography. The absorbance at 319 nm was used to calculate the amount of UV absorbing material released from the spores. Based on the spore numbers in the suspension of the saline extract, we estimated that this material released was 1.4-4.1 pg per spore or 8-24% of the spore weight. Citrinin (and minor amounts of ochratoxin A and some other unidentificable fluorescent compounds) were observed in the filtrate when subjected to thin layer chromatography.

Survey of termite-inhabited soil and mosquito breeding sites in Lucknow, India for potential mycopathogens of Anopheles stephensi.

During a short survey of soil and mosquito breeding sites in Lucknow, India for potential mycopathogen from a period of August-October 1996, 11 species of fungi in 5 genera were isolated using live mosquito larvae as host, Aspergillus flavus, A. fumigatus and Fusarium semitectum were the most frequently isolated species. Other fungi recorded were A. niger, A. ochraceus, A. terreus, A. versicolor, Geotrichum candidum, Penicillium verrucosum, Paecilomyces sp. and Fusarium sp. (Liseola/Elegans complex). Insect cell walls are known to contain chitin, so fungal isolates were tested for their chitinase activity on semi synthetic medium containing colloidal chitin. High chitinolytic activities were observed with A. flavus and A. ochraceus. Chitinase producers can be considered as potential pathogens. However, the higher incidence of F. semitectum could not be explained by inability to utilize chitin.

Varietal differences in accumulation of ochratoxin a in barley and wheat cultivars after inoculation of Penicillium verrucosum

The natural content of ochratoxin A in grain samples of 6 barley, 2 bread wheat and 1 durum wheat cultivars varied from <0.1 to 0.4 ng/g grain. Samples of the analysed cultivars were surface sterilized and kept in humidity chambers at 20°C and water activity (aw) 0.75 or aw 0.85 for 8 days. For both environments, the resulting grain equilibrium water content varied between cultivars of both barley and wheat, attributable to agronomic traits. The samples were then inoculated with Penicillium verrucosum and incubated for up to 23 weeks. With time, all cultivars had increasing ochratoxin A content, with maximum content in different barley cultivars ranging from 34 to 630 ng/g grain for aw 0.75, and 39 to 260 000 ng/g for aw 0.85. Corresponding values for the wheat cultivars were 25 to 2 300 ng/g and 650 to 5 200 ng/g. Significant varietal differences in ochratoxin A accumulation were observed for barley (P < 0.0001), attributable to equilibrium water content, amylose content and natural ochratoxin A, and for wheat (P < 0.0001), attributable to protein content and natural ochratoxin A. Barley ‘SW 1306 95/1203’ and ‘SW 906129 Waxy’, and wheat ‘SW 39103’ accumulated significantly less ochratoxin A than the other cultivars.