Essential oils from oregano and thyme were applied for 24 h as fumigants against the mycelia and spores ofAspergillus flavus, Aspergillus niger and Aspergillus ochraceus, as well as against natural microflora of wheat grains. The minimal inhibitory concentration (MIC) of oregano oil needed to inhibit the mycelial growth of the fungi was 2.0 μl/L, while spores were eradicated following exposure to 2.0 to 2.5 μl/L. The thyme essential oil was less efficient in controlling mycelia and growth was observed even following exposure to 4.0 μl/L. However, the thyme essential oil was fungitoxic to spores (MIC = 3.0 μl/L). In another set of trials the efficacy of the oils and two of their constituents (carvacrol and thymol) in controlling natural microflora of surface-sterilized wheat grain was studied. Of the four materials investigated, only oregano essential oil exhibited fungicidal activity and, following 24 h exposure to 20 μl/L, a significant reduction in the percent of infested grain was observed even after 5 days of incubation on potato dextrose agar. A reduction in the germinability of the grains was evident following exposure to the materials tested. When the fungicidal activity of oregano essential oil was evaluated using grains with different moisture contents (MC), data revealed that the better inhibitory effect was achieved in grain with a high MC. The findings emphasize the toxicity of oregano and thyme essential oils as fumigants against fungi attacking stored grain and strengthen the possibility of using them as an alternative to chemicals for preserving stored grains.

The essential oil of oregano (‘origanum oil’; thymol type oil from Origanum vulgare) inhibited completely the mycelial growth of Aspergillus niger and A. flaous at 400 μg/ml, while A. ochraceus was inhibited at 600 μg/ml. At 700 μg/ml, thyme oil inhibited the mycelial growth of A. flavus and A. niger but not that of A. ochraceus. Fungal spore germination was inhibited by 600 μg/ml of origanum oil and (with the exception of A. ochraceus) by 700 μg/ml of thyme oil. Under aerobic conditions, the essential oils of oregano (250 μg/ml) and thyme (350 μg/ml) inhibited to some extent the growth of Staphylococcus aureus and Salmonella typhimurium. Pseudomonas aeruginosa was not affected by either oregano or thyme oil at concentrations up to 500 μg/ml. The origanum oil was very effective against Campylohacter jejuni and Clostridiurn sporogenes and thyme oil was very effective against C. jejuni. The antagonistic effect of the two oils on Staph. aureus and Salm. typhimuriutn was greatly enhanced when those organisms were incubated in atmospheres of low oxygen tensions

Aflatoxins are carcinogenic metabolites produced by several members of the Aspergillus flavus group in grains and foods. Three genes, ver-1, omt-1, and apa-2, coding for key enzymes and a regulatory factor in aflatoxin biosynthesis, respectively, have been identified, and their DNA sequences have been published. In the present study, three primer pairs, each complementing the coding portion of one of the genes, were generated. DNA extracted from mycelia of five Aspergillus species, four Penicillium species, and two Fusarium species was used as PCR template for each of the primer pairs. DNA extracted from peanut, corn, and three insect species commonly found in stored grains was also tested. Positive results (DNA amplification) were achieved only with DNA of the aflatoxigenic molds Aspergillus parasiticus and A. flavus in all three primer pairs. The detection limit of the PCR was determined by using the primer pairs complementing the omt-1 and ver-1 genes. Sterile corn flour was inoculated separately with six different molds, each at several spore concentrations. Positive results were obtained only after a 24-h incubation in enriched media, with extracts of corn inoculated with A. parasiticus or A. flavus, even at the lowest spore concentration applied (10 2 spores per g). No DNA amplification was observed from corn inoculated with other molds, even at the highest inoculum level (10 6 spores per g). It is concluded that genes involved in the aflatoxin biosynthetic pathway may form the basis for an accurate, sensitive, and specific detection system, using PCR, for aflatoxigenic strains in grains and foods.

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