Natural incidence of cyclopiazonic acid (CPA) in corn from the southern part of China and Southeast Asian countries including Indonesia, Thailand, and the Philippines, was surveyed using a combination of a clean-up procedure consisting of diethyl ether liquid-liquid extraction and HPLC-UV analysis. Out of 99 corn samples analyzed, 15 samples (15%) were contaminated with CPA. The detected concentration of CPA ranged from 27 to 1510 μg/kg, with all CPA positive samples contaminated with aflatoxin B₁ (AFB₁). It was noteworthy that the high level of CPA together with AFB₁ over 1000 μg/kg was found in the samples intended for human consumption from China and the Philippines.

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Cyclopiazonic acid (CPA) contamination in commercial rice koji (molded rice inoculated with koji mold) used for the production of fermented foods in Japan was surveyed using a combination of a clean-up procedure consisting of diethyl ether liquid-liquid extraction and HPLC-UV analysis (quantification limit: 25 μg/kg). Out of 38 koji samples collected from 18 prefectures, one sample was contaminated with CPA at a level of 480 μg/kg. From the CPA contaminated sample, two types of koji mold, one with white colonies and the other with yellowish green colonies, were obtained at equal incidence rates. The presence of CPA in a Japanese commercial rice koji is due to CPA production by the latter type of koji mold.

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Epigallocatechin-3-gallate (EGCG), exhibited protective effect against rubratoxin B (RB)-induced apoptosis in cultured rat hepatocytes. In this study, the mechanism of hepatoprotection by EGCG was investigated. Hepatocytes were concomitantly treated with RB and EGCG and then the expression levels of mitogen-activated protein kinase (MAPK) p38, caspase-3 and caspase-8 genes were measured by reverse transcriptase-polymerase chain reaction (RT-PCR). p38 was induced by RB, indicating that RB-induced apoptosis was mediated by p38 pathway. On the contrary, the elevation of p38 by RB was diminished by EGCG co-treatment. The analysis of caspase-3 mRNA expression showed the same tendency as observed for the p38. To further elucidate, the expression level of mitogen-activated kinase kinase 6 (MKK6) gene, upstream signaling factor of p38, was analyzed by RT-PCR. EGCG also suppressed the expression of MKK6 mRNA. These results suggest that EGCG protects hepatocytes from RB toxicity by the suppression of the gene expression in p38 and caspase pathway at any points examined.

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The interaction of rubratoxin B (RB) with bovine serum albumin (BSA) was investigated. Circular dichroism spectroscopy shows the interaction of RB with BSA as conformational change of BSA. Fluorescence quenching study suggests that RB binds to BSA at a few sites. At low concentration of RB, RB firstly binds to BSA at any binding sites far from Trp residues. By increasing RB concentration, RB binds to BSA at any binding sites near Trp residues.

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The effect of industrial processing on deoxynivalenol (DON) and nivalenol (NIV) was examined in naturally contaminated wheat grains and their milling products (flour). A total 160 samples (80 samples each of wheat grains and flour) were obtained from milling companies. Among them, a pair of 20-lot samples of wheat grain and flour was divided into 4 different uses; home use, cake, noodles, and bread.
The toxins were extracted with acetonitril- water (85:15), and passed through a multi-function column for clean-up. After their trimethylsilyl derivatization, quantitative determination of the toxins was carried out by gas chromatography-mass spectrometry in the selected ion monitoring mode.
The concentration of DON and NIV in wheat grains was ND-2,450 ng/g and ND-174 ng/g, respectively. In the processed products, it was ND-1,620 ng/g for DON and ND-20 ng/g for NIV. The mean and range of reduced DON and NIV rates after processing was 74% (0-97%) and 63% (22-91%), respectively.

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To improve an animal model used in toxicological studies, we investigated the effects of genetics, sex, and age on rubratoxin B toxicity in mice. Treatment with rubratoxin B for 24 h increased serum alanine aminotransferase (ALT) activity, a common indicator of hepatic injury, in C3H/He, BALB/c, DBA/2, and C57BL/6 mice. The increase in serum ALT levels was most pronounced in rubratoxin B-treated C3H/He mice, as was the decrease in serum glucose concentration. In addition, these effects were greater in C3H/He male mice than female. Serum ALT activities were more elevated in rubratoxin B-exposed 9- and 12-week-old animals than that of 6-week-olds. The effect of rubratoxin B on the serum glucose concentration was greatest in 9-week-old male C3H/He mice. Therefore, our findings show that the genetics, sex, and age of the mice treated all contributed to the toxicity of rubratoxin B and that 9-week-old male C3H/He mice were the most susceptible of the animals we tested. Moreover, rubratoxin B-exposed mice had fatty livers and increased levels of triglycerides (the typical lipid involved in the development of fatty liver).

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The provisional standard of 1.1 mg/kg for deoxynivalenol (DON) in wheat was determined by Japanese government in 2002. Therefore, the endpoint in our research must be changed form disease severity to mycotoxin contamination. In this review, I proposed the following strategy for control of Fusarium head blight and mycotoxin contamination.
i) NIV chemotypes of F. graminearum were widely distributed in western part of Japan in 2002 and the isolation frequency was higher than that of DON chemotypes, and moreover, their virulence would be considerably high. Therefore, control of NIV in addition to DON is needed in Japan at least in the western part, and higher level of FHB resistance should be aimed for in wheat and barley breeding than ever.
ii) We found that seven chemicals; metoconazole, tebuconazole, captan, thiofanate-methyl, oxine-copper, copper-hydroxide and phosphorous acid would control DON and NIV. On the contrary, treatment of azoxystrobin significantly increased the level of DON and NIV compared to the control plots, even though it reduced disease severity of FHB. These results suggest that new fungicide evaluation system based on efficacy for mycotoxin contamination should be introduced.
iii) We demonstrated that sieve sorting and gravity sorting were effective to reduce DON and NIV level. In a country elevator, we need more simple ELISA system to check DON and NIV.
iv) In Japanese wheat production, non-till or minimum tillage is not essential and area of a wheat field is relatively small. Therefore, cultural practice to decrease residue is practical options. We are going to test cultural practices of top soil dressing, burning, up-cut rotary and carry out to decrease the rice residue which is primary inoculum source of Fusarium head blight.
v) Despite the importance of the disease, there are still unanswered questions concerning the epidemiology and disease spread. How far can the ascospore inoculum be moved from an infected field? How is a genetic diversity of Fusarium graminearum in a field? These information are key for rational management techniques. We investigated spatial distribution of Fusarium head blight and mycotoxin contamination in naturally infested field. The SSR (simple sequence repeats) markers that could clearly and reliably detect DNA polymorphism among Japanese isolates of Fusarium graminearum were developed. Molecular epidemiology using SSR markers could be a new approach.

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[in Japanese]

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Current status of taxonomy of Fusarium species causing head blight of wheat and barley, especially on the F. graminearum complex, is commented based on the published papers. Two distinct groups or populations have been known among the F. graminearum complex, and the Group 1 population of F. graminearum, causing mainly crown rot of wheat and so on, was recently described as F. pseudograminearum (teleomorph: Gibberella coronicola) based on comparative morphology, molecular phylogenetic analyses and cross experiments among the strains. Phylogenetic structures of the Group 2 population of F. graminearum were also studied and strains of the Group 2 population from various geographic origins were revealed to be consisted of many distinct phylogenetic lineages. Because differentiation of these lineages based on phenotypic characters was difficult, they have been designated with numbers from 1 to 9. Recently eight lineages were described as eight distinct new species based on species-specific fixed nucleotides characters, in addition to F. graminearum s. str., corresponding to the lineage 7.

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Fusarium head blight (FHB) or scab is a significant fungal disease of wheat and barley, imposing a threat to the worlds' food supply. A major causal agent is the ascomycete Fusarium graminearum Schwabe (teleomorph: Gibberella zeae (Schwein.) Petch). In addition to quantitative losses, F. graminearum also causes a reduction in grain quality due to the production of mycotoxins such as deoxynivalenol, nivalenol and zearalenon. These pose a serious hazard to human and animal health. Recent genomic analyses are elucidating many aspects of this fungus.

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Risk analysis is a framework developed and used in the European Union, USA and international organizations for addressing food safety issues. Codex establishes maximum levels for mycotoxins in foods and codes of practice for prevention and reduction of mycotoxin contamination in agricultural products basing on risk analysis principles. EU countries and USA also manage health risks resulting from mycotoxin contamination using the same principles. In order to ensure the safety of agricultural products consumed in Japan, the Ministry of Agriculture, Forestry and Fisheries has introduced risk analysis framework into the preventive control of mycotoxins. Under the risk analysis framework we should base risk management options primarily on risk assessment and scientific data such as myocotoxin surveillance data in agricultural products.

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