[in Japanese]

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The reason that fungi are such prolific elicitors of potent, and often specific, inhibitors of key enzymes and cellular receptors is not known. For the biologist, the most exciting thing about the discovery of a new fungal metabolite is the mystery that accompanies each new metabolite. What benefit does this chemical provide to the fungus? Does this metabolite have biological activity and, if so, what biomolecules does it target? Can it be useful to mankind or is it a toxin that could be harmful to our crops, farm animals, or ourselves? It is possible to roughly estimate the number of fungal metabolites, including those with toxic properties known as mycotoxins. In 1971 Turner cataloged approximately 1200 secondary fungal metabolites produced by approximately 500 species of fungi. In 1983 Turner and Alderidge cataloged 2000 more metabolites produced by approximately 1100 species. Thus, there were approximately two unique secondary metabolites/fungal species. In 1991, Hawksworth estimated that there were 69, 000 known fungal species and that these represented about 5% of the total known species in the world, which Hawksworth estimated at 1, 500, 000. Conservative estimates are on the order of 100, 000 species. Based on the work of Hawksworth and the assumption of two unique secondary metabolites/fungal species, there may be as many as 3, 000, 000 unique secondary fungal metabolites. The conservative estimate would be 200, 000. Between 1971 and 1983 the total number of known secondary fungal metabolites increased from 1, 200 to 3200. Assuming that the rate of discovery (accessible in the published literature) remained at a similar rate then approximately 12, 000 secondary fungal metabolites would be described by the end of 1998 (Fig. 1), less than 0.5% of the 3, 000, 000 or 6% of the 200, 000 unique compounds as estimated above. Clearly, the number of undiscovered secondary metabolites is quite large. Cole and Cox listed approximately 300 secondary fungal metabolites as mycotoxins, about 10% of the secondary fungal metabolites described by Turner and Turner and Alderidge. Thus, one can reason that there are potentially between 20, 000 and 300, 000 unique mycotoxins. The diversity of toxic mechanisms will be equally as great. Given the potentially large number of mycotoxins and the diversity of their mechanisms of action, the potential for extremely complex toxin interactions is also high. Given the past successes and hopes for the discovery of useful products, the immense chemical and mechanistic diversity of fungal metabolites is certainly good news. However, the same fungi that have served so well as chemical factories for producing useful drugs and research tools are also frequent contaminants of the food we eat and the air we breath and, thus, are potential agents of disease. In this sense, fungal metabolites are like a tripled edged sword, capable of cutting in three directions; as research tools for the biochemist, poisons to plants and animals, and therapeutic agents for the treatment of diseases. In this review, recently discovered fungal metabolites that inhibit de novo sphingolipid biosynthesis will be used as an example of this concept.

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Aflatoxins (AFs) are toxic and carcinogenic secondary metabolites produced by certain strains of Aspergillus flavus, A. parasiticus A. nomius and A. tamarii. In order to devise effective methods for preventing aflatoxin contamination of feed and food, elucidation of aflatoxin biosynthetic mechanism by fungi is important. I would like to overview recent topics relating aflatoxin biosynthesis, and then to introduce our recent results about vrdA gene coding versiconal hemiacetal acetate reductase involved in aflatoxin biosynthesis.

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An important and problematic section within Aspergillus is sect. Flavi. Because of their industrial, agronomic and medical importance, the accurate identification and classification is necessary. Traditional methods are based on the morphological characters, but the high degree of morphological similarities make identification difficult, therefore the taxonomy is clouded and controversial [1-4]. In the past, various methods had been used to the identification and classification of the sect. Flavi. But the results of these studies were varied and could not give a clear taxonomic conclusions. Recently, the several molecular attempts have been carried out as tools for studying the phylogenetic relationships, as well as for classifying and identifying the sect. Flavi. For example, based on RAPD analysis, only A. parasiticus and A. sojae were differentiated from each other [5]. The sequence divergence of aflR genes (hybridization patterns) showed that A. oryzae and A. sojae were similar to those of A. parasiticus [6]. Based on PCR-SSCP (single-strand conformation polymorphism) analysis, the strains were divided into four group, A. f lavus, A. oryzae, A. parasiticus/A. sojae, A. tamarii and A. nomius [4]. The analysis of the PCR-RFLP patterns showed except for A. tamarii, there were very similar among the other five species. Based on the analysis of the enzyme electrophoretic patterns, A. flavus, A. parasiticus and A. tamarii showed distinct patterns [2, 7], but A, oryzae was very similar to those of A. flavus, and A. sojae to those of A, parasiticus. While Klich and Mullaney (1987) reported that A. oryzae and A. flavus could be differentiated based on the Sma I digestion patterns using total DNA [8]. Using electrophoretic comparison of enzymes and ubiquinone systems, Yamatoya et al. (1990) found that isolates of A. flavus, A. parasiticus, A. oryzae and A. sojae could be accommodated in two species: A. flavus and A. parasiticus [2]. The substitution rate in mammalian mt DNA was five to ten times higher than that in chromosomal gene [9]. Therefore, it can giving a magnified view of genetic differences among species and allow determination of the relationships among closely related species [10]. Recently, mt DNA analysis by restriction fragment-length polymorphisms (RFLP) has become a useful methods for taxonomy of the fungi [11, 12, 13]. The mt cytochrome b gene was oftenchosen as a phylogenetic probe in birds, mammals and fish [14-19]. To demonstrate the sequences of mt cytochrome b gene can be used for phylogenetic classification and will prove especially valuable identification for fungi, we had firstly reported this gene were very useful and powerful tool in fungi [20]. We had studied major five species of pathogenic Aspergillus [20] and other fungi, for example, Zygomycetes, genus Aspergillus, Penicillium, Neosartorya, and so on (unpublished). These sequences and phylogenetic tree have confirmed it. Here we provide an independent assessment of evolutionary relationships among sect. Flavi, based on nucleotide sequences from mt cytochrome b gene. And using the sequences as a tool for classification and identification.

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Distribution of aflatoxigenic fungi in Japan has been studied extensively because of safety concerns about traditional fermented foods which use A. oryzae or A. sojae for the fermentation. Very limited distributions of these fungi in the warm areas of southwest Japan were reported. Later, Takahashi isolated aflatoxigenic fungi from field soil samples in Kanagawa Prefecture and Goto et al. isolated some aflatoxin producing fungi from silkworm frass including samples collected in the northern part of Japan. However details of these fungi have not been published. Recently, we isolated some aflatoxigenic fungi, all from Aspergillus section Flavi, from acidic tea field soil samples. All aflatoxigenic fungi previously known belonged in A. flavus, A. parasiticus and A. nomius but the recent isolate we reported on produces aflatoxin and cyclopiazonic acid, but is similar morphologically to A. tamarii. Also, aflatoxigenic species from Aspergillus sections other than Flavi have been reported. Important non-aflatoxigenic strains and species from section Flavi include A. oryzae, A. sojae, A. tamarii and A. caelatus.

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Aflatoxins are produced by Aspergillus flavus and Aspergillus parasiticus which principally distribute in the tropical and subtropical regions. In Japan, Manabe et al. showed that the toxigenic fungi were often found in the southern part of Japan such as Kyushu and Okinawa Districts. The fungi infect and contaminate many kinds of agricultural commodities, not only nuts and oil seeds but also other agricultural comodities with aflatoxin. Sugarcane is commonly grown in the tropical and subtropical regions. Tabata et al. showed that aflatoxin was detected in crude sugar (brown sugar) produced in the southernmost part of Japan such as Okinawa although the content of the mycotoxin was low levels of 1.0-1.5 ppb. Therefore, we investigated distribution of aflatoxin-producing fungi in soil and air of sugarcane field as well as harvested sugarcane stem in the southernmost regions. There, we found a variety of aflatoxin-producing fungi including A. parasiticus, and then separated on basis of morphological criteria and mycotoxin producrion.

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