To gain insights into the regulatory mechanism of trichothecene biosynthesis, I worked on molecular genetic studies of Fusarium graminearum in RIKEN and Nagoya University. First, Aspergillus nidulans promoters with different levels of transcription were identified and characterized in F. gramienarum. Second, subcellular localization of Tri6p, a zinc finger protein essential for trichothecene (Tri) gene transcription, was analyzed by using these promoters. When the predicted nuclear localization sequence (NLS) of Tri6 was fused to the EGFP (enhanced green fluorescence protein) gene and transcribed under the control of a strong A. nidulans TEF1α promoter, the EGFP green fluorescence was clearly observed in the nucleus, demonstrating the functionality of the nuclear localization signal. Third, the role of sucrose as an inducer, but not as the carbon source of the medium, in trichothecene production was unambiguously demonstrated. Based on the mode of actions of sucrose and other chemicals that modulate trichothecene production, a new regulatory model of Tri6 expression is now being proposed. Further investigations with molecular and mechanisms of genetic approaches using these tools will clarify the trichothecene biosynthesis regulation.
The dichlorvos-ammonia (DV-AM) method is a simple and sensitive visual method for detecting aflatoxigenic fungi. Since aflatoxin contamination has been frequently reported in ground nuts and tree nuts in many countries, we herein used the DV-AM method for the detection of aflatoxigenic fungi in imported raw nuts and seeds: peanuts from South Africa, macadamia nuts from Australia, pistachio nuts from Iran, macadamia nuts from Kenya, almond nuts from California, U.S., cashew nuts from Viet Nam, hazelnuts from Turkey, pumpkin seeds from China, sunflower seeds from Bulgaria, and walnuts from the U.S. Using the DV-AM method revealed that yellow fungal colonies surrounding the peanuts and the macadamia nuts were changed to red by ammonia treatment. Thin-layer chromatography analyses showed that the red fungi from the peanuts produced mainly aflatoxin B1 and the red fungi from the macadamia nuts produced mainly aflatoxins B1 and G1. Based on their calmodulin gene sequences, the peanuts' fungi were identified as Aspergillus flavus, and those from macadamia nuts from Australia were Aspergillus parasiticus. Aflatoxigenic fungi were not detected in the other nuts and seeds. These results demonstrate that the DV-AM method can be used to detect aflatoxigenic fungi in various imported foods. This method will also be useful for the determination of critial control points for food safety at different stages of the food chain: harvesting, sorting, washing, various food processes, and transporting.
According to the current Fusarium taxonomy, F. asiaticum and F. graminearum sensu stricto are known to cause scabby disease in Japanese wheat and barley. In this study, the previously identified F. roseum KU-117 and eight Gibberella zeae strains were re-classified based on PCR detection using species-specific primers. As a result, the strain KU-117 was identified as F. asiaticum. Of the G. zeae strains, three strains isolated from Aomori were identified as F. graminearum sensu stricto, but the remaining five strains derived from Iwate, Tokyo, Kumamoto, and Nagasaki belonged to F. asiaticum. The RAPD patterns of the G. zeae strains tested showed low genetic diversity. The results obtained here reaffirmed that F. asiaticum and F. graminearum sensu stricto are distributed regionally in Japanese crop fields.