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

Natural occurrence of T-2 toxin in domestic and imported rice

Rice is one of the crops, which are prone to be contaminated with toxigenic fungi and their mycotoxins. This study aimed to investigate the natural occurrence of T-2 toxin in domestic and imported rice in Iran. In a cross-sectional descriptive study in winter 2007, 140 samples of imported rice [125 samples of Thai and 25 samples of Pakistani rice] and 60 samples of Iranian rice were collected from warehouses of canteens of governmental offices in Tehran. After grinding and methanol extraction of the rice samples, the amount of T-2 toxin was measured using a sandwich ELISA. INSTATA statistical software was used for data analysis. All samples of rice were more or less contaminated with T-2 toxin but the amount did not exceed the permissible limit. Mean contamination of domestic and imported rice was 11.2 +/- 2.3 and 13 +/- 2.7 micro g/kg, respectively. Regarding imported rice, mean of contamination was 14.5 +/- 4.6 micro g/kg for the Pakistani rice and 12.6 +/- 2.2 micro g/kg for the Thai rice. There was no significant difference between domestic and imported rice, nor did we find a meaningful difference among Iranian, Pakistani and Thai rice regarding the amount of contamination [P= 0.2]. Although the amount of contamination is less than the safe limit, the extent of natural occurrence of T-2 toxin in rice in Iran indicates that contamination occurs somewhere in the production process. This, in turn, necessitates screening of rice for contamination with mycotoxins from farm to table

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

Preliminary study on virulence of some isolates of entomopathogenic fungi in different developmental stages of Boophilus annulatus in Iran

To evaluate the virulance of 11 isolates of native entomopathogenic fungi as biocontrol agent of Boophilus annulatus, in this study, 4 three months old calves were used for tick rearing. 7 Different developmental stages of the ticks, Boophilus annulatus were inoculated by 10 conidia/ml dilution of the fungal isolates in the presence of control groups. The mortality, egg hatchability and reproductive efficiency were determined in different treatments and control groups and the results were analized statisticaly. Metarhizium anisopliae strains DEMI001 and IRAN437C, Beauveria bassiana strain IRAN403C, and Lecanicillium psalliotae strain IRAN468C were the most virulent strains in comparison with their relative strains and caused 80 -100%, 20 - 80%, 0 - 40% and 0- 40% mortality for engorged females respectively. All 11 tested fungi reduced egg laying capability of the ticks several days before their death. The obtained data showed that the entomopathogenic fungi can affect all developmental stages of Boophilus annulatus, but their efficiency varies considerably according to the fungal species and strains. It is demonstrated for the first time the pathogenic effect of Lecanicillium psalliotae against Boophilus annulatus

Study on the viability of native isolates of Arthrobotrys after passage from sheep's gastrointestinal tract for biological control of gastrointestinal s nematodes

Study on the viability and nematophagousactivity of three native isolates of Arthrobotrys [two A.oligospora and one A. cladodes var. macrolides] afterpassage through sheep's gastrointestinal tract. Field study. Three sheep for each isolate. Three native isolates of Arthrobotrys [two A.oligospora and one A. cladodesvar. macrolide] isolatedfrom soil of different regions of Mazandaran. Each isolatewas cultured on barley and given in equal amounts to threesheep. Four and five days after administration, the faecesof sheep were recultured for isolation of Arthrobotrys sp.and to test their viability and nematophagous activity usingfaecal culture and dung pat bioassay. Analysis of variance [ANOVA]. Conidia of three native isolates of Arthrobotryswere reisolated from faeces of infected animals which kepttheir viability and showed 79.75 - 82.26% and 78.79-89.27% nematophagous activity in faecal culture and dungpat bioassay, respectively. These new isolates are significantlyable to reduce the number of Haemonchus contortusinfective larvae through oral administration and can beconsidered as an alternative for chemotherapy

[Biological Control of Haemonchus contortus by Nematophagous Fungi: Arthrobotrys oligospora, Duddingtonia flagrans and Haptocillium sphaerosporum]

Objective: Biological control of gastrointestinal nematodes of ruminants

Design: Case-control study

Samples: A total of 50 sheep feces naturally infected by Haemonchus contortus ova

Procedure: Arthrobotrys oligospora [111.37 and 251.83], Daddingtonia flagrans [583.91] and Haptocillium sphaerosporum [381.84] were obtained and the nematophagous activity of these isolates was studied after addition of 8000, 20000 and 100000 conidia to 1 gram of fecal samples containing 70 ova of Haemonchus contortus per each petridish. All of the samples were incubated at 25-27°C for 8 days and then, the nematophagous effect of fungal isolates were determined after calculation of third staged larval reduction using Berman method

Statistical analysis: One-way ANOVA and complementary method of Tukey were used

Results: Study of nematophagous effects of 8000 conidia of all above-mentioned fungi and 20000 conidia of A. oligospora [251.82] on the third stage larvae of H. contortus showed that there was not any significant difference as compared with the control groups. But for 20000 conidia of A. oligospora [111.37] and D. flagrans [583.91] and also 100000 conidia for all above-mentioned fungi, significant reduction in larvae of H. contortus was observed as compared with the control groups. In H. sphaerosporum, the percentage of larvae reduction for 8000 and 20000 conidia was determined as 21.46% and 48.99%, respectively. But, the increasion of conidia to 100.000 caused only 42.28% reduction in infective larvae so above-mentioned fungus can not function as an effective agent in biological control of H. contortus

Clinical implications: The present study showed that we can control gastrointestinal nematodes by use of nematode-trapping fungi, in suitable conditions along with chemical treatment.