Polyacrylamide Gel Disc Electrophoresis of Alkaline Proteinases from Aspergillus Species

Ciliatine (2-aminoethylphosphonic acid) (76 mg) was isolated from 72 g of lipids of the oyster with a combination of ion exchange chromatographic techniques and was identified from the results of elementary analysis, infrared spectrum, and chromatographic behaviors. The phosphonic acid was also detected in hydrolysates of a chloroform-methanol insoluble fraction of the oyster. It has been demonstrated that the oyster contains high concentration of ciliatine.     

Influence of near null magnetic field on in vitro growth of potato and wild Solanum species

The influence of near null magnetic field on in vitro growth of different cultures of potato and related Solanum species was investigated for various exposure times and dates. Potato (Solanum tuberosum L. cv. Désirée) in vitro cultures of shoot tips or nodal segments were used. Three different exposure periods revealed either stimulation or inhibition of root, stem, or leaf in vitro growth after 14 or 28 days of exposure. In one experiment the significant stimulation of leaf growth was also demonstrated at biochemical level, the quantity of chlorophyll a and b and carotenoids increasing more than two-fold. For the wild species Solanum chacoense, S. microdontum, and S. verrucosum, standardized in vitro cultures of nodal stem segments were used. Root and stem growth was either stimulated or slightly inhibited after 9 days exposure to near null magnetic field. Callus cultures obtained from potato dihaploid line 120/19 were maintained in near null magnetic field in 2 different months. For these experiments as well as for Solanum verrucosum, callus cultures recorded either slight inhibition or no effect on fresh weight. For all experiments significant growth variation was brought about only when geomagnetic activity (AP index) showed variations at the beginning of in vitro growth and when the explant had at least one meristematic tissue. Moreover longer maintenance in near null magnetic field, 28 days as compared to 14 days or the controls, can also make a difference in plant growth in response to geomagnetic field variations when static component was reduced to zero value. These results of in vitro plant growth stimulation by variable component of geomagnetic field also sustain the so-called seasonal "window" effect.     

Comprehensive proteomic analysis of Penicillium verrucosum

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).     

Influence of carbon and nitrogen sources on ochratoxin A production by two species of Penicillium isolated from poultry feeds

Mycotoxins are natural, secondary metabolites produced by fungi on agricultural commodities in the field and during storage under a wide range of climatic conditions. The ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by several species of Aspergillus and Penicillium. In this study, the influence of carbon and nitrogen sources on ochratoxigenic Penicillium species was assessed. The ochratoxigenic Penicillium species were isolated from poultry feed samples of Andhra Pradesh, India. The isolated ochratoxigenic Penicillium species were identified, screened and characterised as OTA producers by high performance thin layer chromatography (HPTLC) and confirmed by high performance liquid chromatography (HPLC). This experiment was carried out using Czapak yeast Autolysate (CYA) medium with different carbon (C) and nitrogen (N) sources at pH 6.5 and incubated at 25 ± 2°C under dark condition. Maximum OTA production was recorded in the presence of D-glucose followed by D-galactose and D-lactose as carbon sources. Similarly, the maximum amount of OTA production was observed in thiourea followed by potassium nitrate as nitrogen source. However, OTA production, final pH of the medium, and mycelial yield and OTA production of both the species of Penicillium varied with C and N present in the medium. The kinetics of the both species of Penicillium was observed for 30 days at an interval of three days. The maximum amount of OTA was detected by 12 and 15 days incubation periods for Penicillium nordicum and Penicillium verrucosum, respectively.     

Stress induction of mycotoxin biosynthesis genes by abiotic factors

Systematic expression analysis of mycotoxin biosynthesis genes by real-time PCR and microarray was carried out to examine the relationship between growth and general expression patterns in relation to single environmental factors such as temperature, water activity (a(w)) and pH and water activity x temperature interactions. For single parameters, one major peak of expression occurred close to optimum growth conditions. However, a second minor peak was observed under suboptimal growth conditions, when intermediate environmental stress was imposed on Aspergillus parasiticus (afl genes), Penicillium verrucosum (ota genes) and Fusarium culmorum (tri genes). This expression profile pattern was more pronounced in relation to changes in temperature and a(w) than to pH. In a two-factorial experimental design with temperature xa(w) regimes, again two peaks of expression were observed for cluster genes after microarray analysis, one close to those giving optimal growth and one under imposed stress conditions. Interestingly, when the activity of single genes of the microarray data were plotted in relation to the two parameters, again a two-peak expression profile became obvious independently for both parameters. Expression of the mycotoxin biosynthesis genes was followed exactly by phenotypic mycotoxin production. This expression profile appears to be generic across the mycotoxigenic fungi examined.     

Ochratoxin A producing Penicillium verrucosum isolates from cereals reveal large AFLP fingerprinting variability

AIMS: To examine if molecular amplified fragment length polymorphism (AFLP) fingerprinting of the only ochratoxin A-producing species in European cereals, Penicillium verrucosum, can be used as a method in hazard analysis using critical control points (HACCP). METHODS AND RESULTS: A total of 321 isolates of P. verrucosum were isolated from ochratoxin A-contaminated cereals from Denmark (oats), UK (wheat and barley) and Sweden (wheat). Of these, 236 produced ochratoxin A as determined by thin layer chromatography; 185 ochratoxin A-producing isolates were selected for AFLP fingerprinting. A total of 138 isolates had unique AFLP patterns, whereas 52 isolates could be allocated to small groups containing from two to four isolates with similar AFLP patterns. A total of 155 clones were found among the 185 P. verrucosum isolates, thus 84% of the isolates may represent different genets of P. verrucosum. As the few isolates that were grouped often came from the same farm, and those groups that contained AFLP-identical isolates from different countries were morphotypically different. On single farms up to 35 clones were found. The few groups of ramets from the same genet indicated that a HACCP approach based on clones may require a very large number of AFLP analysis to work in practice, we recommend basing the HACCP approach on the actual species P. verrucosum. A more detailed characterization should rather be based on the profile of species present at different control points, or analysis of the mycotoxins ochratoxin A and citrinin in the isolates. Examination of 86 isolates with HPLC and diode array detection of P. verrucosum showed that 66% produced ochratoxin A, 87% produced citrinin, 92% produced verrucin and 100% produced verrucolone. CONCLUSIONS: Among 184 ochratoxin A-producing Penicillium verrucosum, 155 clonal lineages were indicated by AFLP fingerprinting, indicating a high genetical diversity, yet the species P. verrucosum is phenotypically distinct and valid. SIGNIFICANCE AND IMPACT OF THE STUDY: AFLP fingerprinting of Penicillium verrucosum indicates that genetic recombination takes place in this fungus.     

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. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evaluation of larvicidal potential of certain insect pathogenic fungi extracts against Anopheles stephensi and Culex quinquefasciatus

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.