Effect of curcumin on aspergillus parasiticus growth and expression of major genes involved in the early and late stages of aflatoxin biosynthesis

The effect of curcumin as a natural safe compound with different biological activities was examined on fungal growth and aflatoxin production in Aspergillus parasiticus NRRL 2999. The fungus was cultured in presence of serial two-fold concentrations of curcumin [125-2000 micro g/ml] in yeast extract sucrose broth for 3 days at 28°C. Mycelia dry weight was determined as an index of fungal growth, while aflatoxin production was assessed by high performance liquid chromatography [HPLC]. The expression of ver-1, nor-1, pksA, omtA and aflR genes in aflatoxin biosynthetic pathway was evaluated by real time PCR. Curcumin strongly inhibited aflatoxin B1 production in the range of 26.6 to 94.9% by serial two-fold concentrations from 125 to 2000 micro g/ml. Fungal growth was also inhibited by the compound in the range of 34.0 to 60.8%. Analysis of the expression of aflatoxin pathway genes by real time PCR showed that curcumin inhibited the expression of ver-1, nor-1, pksA, omtA and aflR genes at concentrations of 250 and 1000 micro g/ml. In concentration of 1000 micro g/ml, gene expression was reduced by 31.3%, 44.6%, 57.1% 110.9% and 286.7% accordingly. Reduction in the expression of aflatoxin biosynthesis genes was significant only for aflR. In ferric reducing ability of plasma [FRAP] assay, curcumin showed strong antioxidant activity at all concentrations tested. Curcumin may be employed successfully as a good candidate in controlling of toxigenic fungal growth on food and feed and subsequent contamination with aflatoxins in practice

Molecular identification of antagonistic bacteria from Tehran soils and evaluation of their inhibitory activities toward pathogenic fungi

To find antagonistic bacteria with potential antifungal activity against some pathogenic fungi, including Aspergillus niger, A. flavus, Fusarium moniliforme and Penicillium marneffei, a total of 148 agricultural soil samples from different sites of Tehran were examined. Antagonistic soils were selected by screening against A niger on glucose-yeast extract [GY] agar using a visual agar plate assay method. All growing bacteria were examined for antifungal activity, and antagonistic bacteria identified based on 16S rRNA sequence analysis. Among a total number of 97 bacteria isolated form inhibitory soils [36 samples], 16 bacteria were reported as strong growth inhibitors in co-cultures on GY agar with all tested fungi at variable degrees. Fungal growth inhibitory bacteria were cultured against all fungi and growth inhibition was measured and analyzed between test and control groups by statistical analysis [ANOVA]. Molecular identification of antagonistic bacteria indicated that most bacterial isolates belonged to the genus Bacillus [81.25%], including B. subtilis [5 isolates], B. amyloliquefaciens [6 isolates] and B. valismortis [2 isolates], followed by one isolate [6.25%] from each Streptomyces sp., Pseudomonas chlororaphis and Acinetobacter baumannii. Based on the visual plate assay results, total fungal growth inhibition of all bacteria was reported in the range of 13.2 to 68.3%. P. chlororaphis SI 05 was reported as the most potent antagonistic bacterium which inhibited the growth of A. niger by 68.3%, followed by F. moniliforme [66.4%], A. flavus [64.7%] mdR marneffei [57.1%].P. chlororaphis and some other inhibitory bacteria reported in the present study, they may be considered not only as a rich source of useful metabolites with potential application in antifungal drug discovery, but also as potential candidates for biological control programs

Application of PCR-RFLP to rapid identification of the main pathogenic dermatophytes from clinical specimens

In the present study, a PCR-RFLP based molecular technique was designed to rapid identification of dermatophytes in clinical specimens. Skin scrapings obtained from human cases suspected to dermatophytosis were studied in order to identify involved etiological fungi. In this experimental study, the specimens [skin scrapings] of patients referred to Mycology Department of Pasteur Institute of Iran were inoculated on Petri dishes contained selective agar for pathogenic fungi [SAPF] and incubated at 25°C until visible growth of fungal colonies. The colonies were examined for standard morphological characteristics after visible growth on the agar medium. A small portion of each fungal colony was further studied by restriction fragment length polymorphism [RFLP] analysis of the PCR-amplified internal transcribed spacer [ITS] region of ribosomal DNA [rDNA]. PCR amplicons were electrophoresed on 2% agarose gel after digesting by different restriction enzymes including MvaI, HinfI and Hae III. Among 160 clinical samples examined, 6 dermatophyte species including Trichophyton mentagrophytes, T. rubrum, T. verrucosum, T. tonsurans, Microsporum canis and Epidermophyton floccosum were finally identified based on the colony morphology and microscopic criteria. Specific PCR products and RFLP patterns for MvaI, HinfI and Hae III enzymes allowed the rapid identification and reliable differentiation of isolated dermatophytes at the genus or species level for 5-10 day-old colonies. The results showed that PCR-RFLP analysis of the ITS region of rDNA is a rapid and reliable tool which allows identification of major pathogenic dermatophytes isolated in this study at species level in young 5-10 day-old colonies