Fusarium species are common filamentous fungi, and are distributed worldwide in crop fields. Some of the species are known to produce mycotoxins such as trichothecenes, zearalenone, and fumonisins, which are responsible for mycotoxicoses in humans and animals. Since 1980, I have been studying the chemical characteristics of selected Fusarium species, along with plant and murine pathogenicity caused by Fusarium crookwellense and Fusarium solani, respectively. In this review, I outline my experimental Fusarium studies, including some experiences during those studies.

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  The major mycotoxins such as aflatoxins, patulin, ochratoxins, citrinin and fumonisins, zearalenone were researched for food safety. Analytical methods for aflatoxins, patulin, ochratoxins, citrinin and fumonisins were developed. Each method consists of quantification and confi rmation, and has good performance in recovery, sensitivity, repeatability, and selectivity. With these methods, mycotoxin contamination in food and foodstuffs were studied. Aflatoxins were found in nuts, cereals, spices, beans and dairy products. Some samples contained more than 10 μg/kg of afl atoxin B₁ , the regulatory level then in Japan. Aflatoxin contamination in buckwheat, coix seed, crude sugar, white pepper and red pepper were found at the first time in Japan. Physical, chemical and biological degradation of aflatoxins was investigated. Pure aflatoxins were reduced by some physical or chemical procedure. But detoxification of aflatoxins in foods was quite difficult because of the stability of aflatoxins in foods. Patulin was found in some apple juice. It was the first finding of patulin contamination in Japan. Patulin was also found in domestic apples. This fact revealed that patulin contamination occurs in Japan. Ochratoxins were found in cereals, coffee, cacao, and fruit products. Most levels of ochratoxins were less than 1 μg/kg. Citrinin was found in cereals, and some of them were co-contaminated with ochratoxin. Developing analytical methods, examinations, and regulations for mycotoxins are considered to be important for food safety.

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  The fate of Fusarium mycotoxin nivalenol during milling of a Japanese wheat cultivar was investigated. Grain samples with two distinct nivalenol levels were test-milled to produce six flour fractions (Breaking flours: 1B, 2B, and 3B, Middling flours: 1M, 2M, and 3M) and two outer layer fractions (bran and shorts). Patent flour for human consumption was made from 1B, 1M, 2B, and 2M, while low-grade flour was made from 3B and 3M. These four samples; Patent flour, low-grade flour, bran and shorts were analyzed for the content of nivalenol by HPLC-UV. Two samples showed similar patterns of nivalenol distribution in milling fractions.

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  Tri13 encodes a cytochrome P450 monooxygenase that catalyzes C-4 hydroxylation of some intermediates in the biosynthesis of type A and type B trichothecenes. This work demonstrated that Tri13 (FsTri13) of Fusarium sporotrichioides, a typical type A trichothecene producer, can participate in the biosynthesis of type B trichothecenes. When FsTri13 was expressed in Fusarium graminearum that produces 3-acetyldeoxynivalenol (3-ADON) as an end product, 3-acetylnivalenol (3-ANIV) was newly detected in the liquid medium. Although 3-ANIV can easily be prepared enzymatically from nivalenol (NIV) by using recombinant trichothecene 3-O-acetyltransferase (TRI101), there has been no conclusive report on the production of 3-ANIV in cultures of Fusarium strains. Possible biosynthetic pathway for production of 3-ANIV was suggested with regard to the function of Tri8, which is responsible for deacetylation of the side-chain O-acetyls in 3-ADON biosynthesis.

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  Economically devastating outbreaks and epidemics of Fusarium head blight (FHB) or scab of wheat and barley have occurred worldwide over the past two decades. Although the primary etiological agent of FHB was thought to comprise a single panmictic species, Fusarium graminearum, a series of studies we conducted over the past decade, employing genealogical concordance/discordance phylogenetic species recognition (GCPSR)¹⁾, revealed that this morphospecies comprises at least 16 phylogenetically distinct species (referred to hereafter as the F. graminearum species complex = FGSC). Results of a multilocus molecular phylogeny, based on maximum parsimony and maximum likelihood analyses of 12 combined genes comprising 16.3 kb of aligned DNA sequence data, suggest that the different species groups within the FGSC radiated in Asia, North America, South America, Australia and/or Africa. The significant biogeographic structure of these lineages, together with evidence of disjunct species in Asia and North America, are consistent with widespread allopatric speciation within the FGSC. In contrast to the results obtained using GCPSR, morphological species recognition using conidial characters and colony morphology was only able to distinguish 6 species and 3 species groups among the 16 species within the FGSC, highlighting the need for sensitive molecular diagnostic tools to facilitate species identifi cation. A validated multilocus genotyping assay was developed to address the need for species determination and trichothecene toxin chemotype prediction, and this assay has been extraordinarily useful in the discovery of novel FGSC species represented in our global FHB surveys. Ongoing molecular and phenotypic analyses are being conducted to elucidate the full spectrum of FHB pathogen diversity, their trichothecene toxin potential and biogeographic distribution. Increased understanding of the distribution and agricultural significance of variation within the FGSC is needed for the development of novel disease and mycotoxin control strategies, including improvements in agricultural biosecurity designed to limit the introduction and spread of non-indigenous FHB pathogens.

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  Quantitative NMR (qNMR) was successfully applied to determine the purities of commercial reagents of trichothecenes, deoxynivalenol (DON), 3-acetyl-DON (3-Ac-DON), 15-acetyl-DON (15-Ac-DON), nivalenol (NIV), T-2 toxin, which are widely used as the analytical standards for the quantitation. Since qNMR is based on the fact that the signal intensity of a given NMR resonance is directly proportional to the molar amount of that nucleus in the sample, the purities were calculated from the ratio of the proton signal intensities between the sample and 1,4-bis(trimethylsilyl)bezene-d₄ (1,4-BTMSB-d₄) which is developed as one of qNMR reference materials with traceability to the International System of Units (SI). As result, the metrological traceable purities of the reagent products were determined as 82.9-98.7% less than the labeled values by manufacturers. To our knowledge, this is first report to determine the metrological traceable purities of trichothecene reagent products. The purity test using qNMR will indirectly improve the reliability of the official analytical method and the quantitative values of trichothecenes in food.

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  In the notice of “The analytical method for total aflatoxins” which has been emitted in August 2011, two kinds of methods were introduced. One is a method using multifunctional column which is applied to cereals, beans and seeds and the other is a method using immunoaffinity column which is applied spices (red pepper, paprika), roasted cereals and samples that cannot be purified by multifunctional column. In this proceeding, I introduce an example and analytical results of a method using multifunctional column.

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  The official method of analysis for the total aflatoxins was notified in august 16, 2011. It shows that the multifunctional column method is applicable to cereals, pulses, nuts and seeds, and the immunoaffinity column method is applicable to spices, processed foods, and the foods which cannot be applied to the multifunctional column method. However, our studies showed that some foods were not applicable to the immunoaffinity column method on the performance criteria for the method of analysis for total aflatoxins to be used by laboratory. In this paper, the applicability of the immunoaffinity column method and its revised method about various foods are mainly described.

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  In 1971, Aflatoxin B₁ (AFB₁) regulation of food was set in Japan and it has not been changed since thin-layer chromatograph method was adopted. But last year, the total aflatoxins of food were regulated and the method was improved. In order to offer the quick and reliable results to clients, it is highly necessary to employ the high-performance liquid chromatograph-mass spectrophotometer(LC-MS/MS), ensure the uniformity of the sample and control high quality. In this paper, we would like to introduce the current situation of total Aflatoxins test.

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  In rodents, ochratoxin A (OTA) induces high incidences of renal adenomas/carcinomas. Since the results of conventional mutagenicity tests have caused controversy, the modes of action remains unknown. In the present study, we have noted the site specificity of OTA. We conducted the reporter gene mutation assay and global gene expression in the cortex (COR) and the outer medulla (OM) in the kidney of gpt delta rats carrying gpt and red/gam (Spi^-) genes, given OTA at a carcinogenic dose. As a result, Spi^- mutant frequencies (MFs), indicating an increase in deletion mutations, but not gpt MFs in the OM were significantly higher than in controls despite the absence of changes in the COR. In addition, to identify genes relating to OTA-induced carcinogenesis, comparison of gene expression changes between the COR and OM in response to OTA was performed. In the comprehensive analysis, up-regulated genes observed in only the OM were as follows: genes associated with DNA double strand break repair, cell cycle progression, G2/M arrest in response to DNA damage, and regulation by p53 or control of p53 function. The overall data suggest that OTA-induced deletion mutations might occur in the process of DNA double strand break repair. The observation of changes in several genes associated with DNA damage might imply that genotoxic mechanisms are involved in OTA-induced renal carcinogenesis.

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  Taxonomy of the main ochratoxin A-producing species belong to Aspergillus section Circumdati (Aspergillus ochraceus group) has been recently revised with the description of new species such as Aspergillus westerdijkiae and A. steynii. Since the high morphological similarity of these species and A. ochraceus makes difficult discrimination among them, molecular analyses such as β-tubulin and other gene sequence were developed to identify OTA-producing fungi in A. ochraceus group into these species mentioned above. Taxonomy of food-borne isolates which were previously identified as OTA-producing A. ochraceus in Japan was re-examined by both traditional and Molecular methods. As a result, 19 of 21 strains were re-identified as A. westerdijkiae, and two strains were re-identified as A. steynii. The molecular methods provide a useful tool accurate discrimination among the most relevant OTA-producing species within sect. Circumdati.

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