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We analysed the levels of Alternaria, Cladosporium, Fusarium and Penicillium verrucosum in grain samples harvested in 2011 and 2012 from conventional and organic farms using qPCR. In general, both Alternaria and Cladosporium occurred in all cereal grains in the highest quantities, followed by P. verrucosum and Fusarium. Alternaria, Cladosporium and P. verrucosum had the highest levels in crop mixtures, barley and rye and lower levels in wheat, while Fusarium levels were the highest in crop mixtures and wheat. The levels of Alternaria and P. verrucosum were higher in organic rye and wheat than conventional grains. Although the level of Fusarium was higher in conventional than organic rye, opposite results were obtained for crop mixtures. A positive correlation was found between Alternaria, Cladosporium and P. verrucosum, indicating that similar factors might affect the distribution of these fungi in grains.

Cereal crop plants are colonized by many fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecene mycotoxins. A multiplex real-time PCR method using TaqMan probes was developed to simultaneously detect and quantify these mycotoxigenic Fusarium, Penicillium and Aspergillus species in cereal grains. Primers and probes used in this method were designed targeting the trichothecene synthase (Tri5) gene in trichothecene-producing Fusarium, rRNA gene in Penicillium verrucosum, and polyketide synthase gene (Pks) in Aspergillus ochraceus. The method was highly specific in detecting fungal species containing these genes and was sensitive, detecting up to 3 pg of genomic DNA. These PCR products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). The method was validated by evaluating sixteen barley culture samples for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi. Among the barley culture samples tested, 9 were positive for Fusarium spp, 5 tested positive for Penicillium spp, and 2 tested positive for Aspergillus spp. Results were confirmed by traditional microbiological methods. These results indicate that DON- and OTA-producing fungi can be detected and quantified in a single reaction tube using this multiplex real-time PCR method. Practical Application: This method would be helpful in detecting and quantifying the mycotoxin producing fungi such as Fusarium, Aspergillus, and Penicillium in cereal grains and cereal-based foods.

BACKGROUND: The use of aromatic plants and their extracts with antimicrobial properties may be compromised in the case of cheese, as some type of fungal starter is needed during its production. Penicillium verrucosum is considered a common cheese spoiler. The aim of this study was to evaluate the innovative use of certain aromatic plants as natural cheese covers in order to prevent mycotoxicogenic fungal growth (P.verrucosum). A collection of 12 essential oils (EOs) was obtained from various aromatic plants by solvent-free microwave extraction technology, and volatile characterisation of the EOs was carried out by gas chromatography/mass spectrometry. RESULTS: The most effective EOs against P.verrucosum were obtained from Anethum graveolens, Hyssopus officinalis and Chamaemelum nobile, yielding 50% inhibition of fungal growth at concentration values lower than 0.02 µ Lm L −1 . All EOs showed high volatile heterogeneity, with α-phellandrene, pinocamphone, isopinocamphone, α-pinene, camphene, 1,8-cineole, carvacrol and trans-anethole being found to be statistically significant in the antifungal model. CONCLUSION: The use of these aromatic plants as natural covers on cheese can satisfactorily inhibit the growth of some mycotoxicogenic fungal spoilers. Among the volatile compounds present, α -a ndβ-phellandrene were confirmed as the most relevant in the inhibition. c � 2012 Society of Chemical Industry

Mycotoxigenic molds can grow on the surface of sausages and can influence their safety for consumption. The aim of this study was to investigate the presence of ochratoxin A (OTA) and ochratoxin-producing molds on the surface of traditional sausages in areas in Northern Italy, including the Central Alps, the Prealps, the Apennines, the Central Padana Valley and the Northeast. We then sought methods for preventing the growth of ochratoxigenic molds and for eliminating them. Our results demonstrated that 24% of the sampled sausages were OTA-positive, and Penicillium verrucosum, Penicillium nordicum and Aspergillus ochraceus were the main ochratoxigenic molds isolated. Brushing and washing treatments reduced the OTA concentrations on sausage casings to below the Italian law limit (

Fungal metabolites are attracting attention as potential microbial insecticides, and they are anticipated to overcome the problems of pesticide resistance and environmental pollution that are associated with the indiscriminate use of conventional synthetic insecticides. The relative bioefficacies of selected fungal pathogens, Aspergillus flavus, A. niger, A. parasiticus, Fusarium sporotrichoides and Penicillium verrucosum were observed against Anopheles stephensi and Culex quinquefasciatus larvae. A. flavus demonstrated the greatest bioefficacy with 50% lethal concentration (LC50) values of 9.54 and 10.98 ppm against Anopheles stephensi and Culex quinquefasciatus larvae, respectively, after 24‐h exposure. The bioefficacy of A. flavus increased in both species with an exposure time of 48 h, with LC50 values of 7.26 and 8.55 ppm, respectively.

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.

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).

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.

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.

Fresh catfish (Clarias gariepinus) were subjected to different concentrations of sodium benzoate or potassium sorbate and smoked traditionally before evaluation for microbiological, chemical and organoleptic characteristics during ambient tropical storage. Unsmoked fish samples showed diverse microflora (Enterobacter, Escherichia, Serratia, Bacillus, Staphylococcus, Streptococcus, Penicillium, Aspergillus and Achlya genera) while smoked samples were dominated by Gram-positive bacterial flora (Bacillus, Staphylococcus and Streptococcus) and spoilage moulds (Penicillium verrucosum, Aspergillus flavus and Achlya spp.). Significant reduction in microbial population occurred in most samples following smoking with samples subjected to 0.4% (w/v) potassium sorbate showing the lowest microbial load and maximum shelf-stability. However, marked microbial increase occurred after day 4 of storage in control and benzoate-treated samples. Changes in pH were marginal but decreased after day 12 of storage. Moisture content decreased sharply after smoking and remained low after day 4 of storage. Overall, potassium sorbate treatment (0.4% w/v) was most effective in controlling microbial quality and extended the shelf-life of the samples by 8 d.

Autoclaved barley and wheat samples were separately inoculated with Aspergillus alutaceus (formerly called A. ochraceus Wilhelm NRRL-3174) and Penicillium verrucosum Dierckx and incubated at 28C for 7, 15 and 30 days. Ochratoxin A (OA), fungal growth (glucosamine concentration), changes in the content of starch, lipids and protein, and the amino acid profile were estimated. OA production of P. verrucosum on both barley and wheat increased significantly over time (P < 0.05 and P < 0.01) with the values being 56 μg/g and 109 μg/g, respectively (after 30 days of incubation). OA production was significantly higher (P < 0.01) for A. alutaceus on wheat (79μg/g) compared with that on barley (38 μg/g). Furthermore, wheat supported growth of both fungi better than barley (P < 0.01). The principal changes in cereal constituents induced by the ochratoxigenic fungi were a marked decrease in the lipids (a minor component) in wheat and barley (P < 0.05), a modest decrease in their content of starch (P < 0.05), an enhanced concentration of protein in wheat (P < 0.06 and 0.01 with the two fungal species), and a change in the relative concentrations of some of the amino acids in wheat (P < 0.05). These results demonstrate that under certain conditions relatively large amounts of OA can be produced in grains with correspondingly small effects on the content of the starch and protein.