Fusarium species are plant pathogenic fungi commonly found in the field. Wet and temperate weather during the growth of grain plants often results in diseases caused by several Fusarium species, which cause two forms of agricultural damage: a reduction in harvest (shriveled grains) and a threat to food safety (contamination of grains by mycotoxins). Since 2001, I have been involved in the development of analytical methods for major Fusarium toxins that can potentially pollute crop grains (especially rice and wheat) and the investigation of the retention of these toxins during processing, such as milling. In this review, I will present two topics; “Detection of fumonisins in rice” and “Retention of deoxynivalenol and nivalenol during milling of wheat”.
Species of the genus Fusarium are well-researched in many fields, and a commonly problem by researchers interested in Fusarium species is the probable taxonomic system and the identification method of this genus. The traditional taxonomic system for fungi has been proposed based on the mainly morphological species concept, including the genus Fusarium. Recently, many researchers have applied molecular markers to examine the taxonomy and identification of Fusarium species. This review shows some recent findings from our studies about molecular phylogeny and identification of Fusarium species based on analyses with nucleotide sequences. First, the genetic markers were evaluated for identifying Fusarium isolates by calculation of the homologies with pairwise comparison of all tested strains, and of the ratio of nucleotide substitution rate. It was suggested that aminoadipate reductase gene (lys2) is notionally the most appropriate genetic marker for identifying isolates among the six genes examined. Second, actual identification of food-borne isolates of the genus Fusarium based on the nucleotide sequence homology was performed, and the results were evaluated. In terms of accuracy and ease, b-tubulin gene, not lys2, is the most useful genetic maker among the six genes examined. Finally, the genetic markers were evaluated for the phylogenetic analysis of Fusarium species. It was suggested the lys 2 have a singular evolutionary history than other genes. To obtain a reliable phylogeny for Fusarium species, the lys2 sequences were excluded from the dataset, and the species tree was inferred. The reliable Fusarium species tree was reconstructed, and some interesting relationships were newly described.
Maize is one of the most important staple cereal grains grown and consumed in Nigeria. One hundred and four maize samples destined for human consumption were collected from thirteen popular different markets during a survey of three agro-ecological zones (AEZ) and levels of zearalenone (ZEA) and T-2 toxin (T-2) were determined by enzyme-linked immunosorbent assay (ELISA). Levels of ZEA contamination of maize samples ranged from < 50 μg/kg to 196 μg/kg. Although, ZEA was detected in 37% of the total maize samples, only 3.8% of these maize samples exceeded the EU maximum recommended limit for ZEA in unprocessed cereals of 100 μg/kg. Also, T-2 toxin was detected in 36% of the total maize samples, and the range of contamination was < 7.5 to 29 μg/kg. At present, there is no EU maximum tolerable limit for this toxin in unprocessed cereals. Eighteen samples constituting 17% of the total samples were found to be contaminated with the two toxins. On the distribution of these toxins on AEZ, the Derived Savanna (DS) zone had more positive samples of 50 and 38% for ZEA and T-2 respectively than the other two AEZ, though more samples were collected from Southern Guinea Savanna (SGS).
14-3-3 family proteins are involved in various signal transduction pathways. We have previously demonstrated that 14-3-3β/fourteen-three-three beta interactant 1 (FBI1) /specific protein 3 (Sp3) transcription factor/histone deacetylase1 (HDAC1) complex suppresses the mitogen-activated protein kinase phosphatase 1 (MKP-1) gene expression, which negatively regulates the MAP kinase signaling cascade, in aflatoxin B1 (AFB1)-induced rat hepatocellular carcinoma (rHCC) K2 cells and finally promotes metastasis and tumorigenicity. In this study, to further elucidate the malignant conversion mechanism of K2 cells, we performed microarray analysis to identify the genes controlled by 14-3-3β/FBI1/Sp3/HDAC1 complex. The data showed that secreted protein, acidic, and rich in cysteine (SPARC) was identified as one of the downregulated target genes. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) analysis revealed that 14-3-3β/FBI1/Sp3/HDAC1 complex bound to GC box in the SPARC promoter region and repressed the transcription. Furthermore, enforced expression of SPARC in K2 cells inhibited colony formation in semi-solid medium. Thus, these results provide the evidence that downregulation of SPARC by the 14-3-3β/FBI1/Sp3/ HDAC1 complex is involved in the malignant conversion of K2 cells.
Trichothecenes are mycotoxins produced by Fusarium and other genera that can cause serious health problems in humans and livestock. We previously reported 22 trichothecene resistance genes in Saccharomyces cerevisiae, whose deletion conferred high sensitivity to trichothecenes. In this study, we constructed various multiple gene deletion mutants of these resistance genes to develop a sensitive yeast bioassay system for trichothecenes. Among the double and triple null mutants, pdr5Δ erg6Δ rpb4Δ cells showed high sensitivity to T-2 toxin and deoxynivalenol under optimized culture conditions (IC50 values of 1.5 ng/mL and 1.5 μg/mL, respectively). The disc diffusion test also confirmed the high sensitivity of this mutant to the trichothecenes. This detection system was more sensitive than any other yeast bioassay previously developed. The pdr5Δ erg6Δ rpb4Δ cells detected 1.0 ppm deoxynivalenol in wheat flour and 1.1 ppm deoxynivalenol in wheat grain, which are the advisory level for deoxynivalenol in final wheat products in US and the provisional level for wheat grain in Japan, respectively. Here, we report an easy, inexpensive, and highly sensitive yeast bioassay system to detect trichothecenes.
The Kuro-Koji mold has been used in fermentation to produce a Japanese traditional spirit in southern part of Japan and the mold is very close to Aspergillus niger morphologically and physiologically. Then, they sometimes have been considered as synonym. Mycotoxin production and phylogenic relationships between them were studied. Some of the A. niger strains produced ochratoxins and most of the strains were producer of fumonisin B2 whereas none of the Kuro-Koji mold strains synthesized the mycotoxins. Genetic analysis including cytochrome b DNA gene showed DNA types of the Kuro-Koji mold can be clearly distinguished from those of the mycotoxigenic strains of A. niger tested. Our study suggested that the Kuro-Koji mold as the producer of shouchu is a distinct genetic clade separated from A. niger although they are morphologically and physiologically very close.
To assess the position of Kuro-Koji molds in black Aspergillus, we performed sequence analysis of approximately 2,500 nucleotides of partial gene fragments, such as histone 3 , on a total of 57 Aspergillus strains, including A. kawachii NBRC 4308, 12 Kuro-Koji molds isolated from awamori breweries in Japan. Sequence results showed that all black Aspergillus strains could be classified into 3 types. All 12 Kuro-Koji molds isolated from awamori breweries were classified into same type which includes A. kawachii NBRC 4308, thus we concluded this type represents the industrial Kuro-Koji molds. We also found these strains lack the An15g07920 gene which is required for ochratoxin A biosynthesis in black Aspergillus. Based on industrial importance and the safety of Kuro-Koji molds, we propose to classify these strains as A. luchuensis, as initially reported by Dr. Inui.
Fumonisins are carcinogenic mycotoxin. More than 20 analogs of fumonisin were found. It has been considered that fumonisins are produced by Fusarium spices and frequently found from corn and corn products. However, recently it was found that Aspergillus niger has a gene cluster for fumonisins. So fumonisin producing ability of A. niger was evaluated. As the result, it was revealed that A. niger produce fumonisin B2 and B4, and not produce fumonisin B1 nor B3. And then, fumonisin B2 and B4, not B1, were found from grape products and coffee. These findings suggested that fumonisins were naturally occurred by A. niger. Further evolution of research on fumonisin contamination by A. niger was expected, with the solution of the problems in analysis.
The fluorescence fingerprint, also known as the excitation-emission matrix （EEM）, is a set of fluorescence spectra acquired at consecutive excitation wavelengths to create a three-dimensional diagram. The pattern of this diagram, rather like the actual fingerprint, is unique for each measured sample, containing abundant information about the constituents making up the sample. Therefore, fine distinctions can be made between samples, which would otherwise be indistinguishable. Detection of mycotoxins in wheat and nutmeg using this technology are introduced in this paper.
Matrix assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF MS） is a new powerful tool to identify the profiles of the ribosome proteins derived from microorganisms. By comparison between the fingerprints of ribosome proteins of microorganism and those of the standard strain library, we can easily identify the species of microorganisms in the clinical sample. This new MS method is more specific and rapid than the conventional method. In our laboratory, we have been directly identified microorganism and yeast in a colony on the solid medium from the clinical sample （blood, urine, feces, sputum, etc.） by the MALDI-TOF MS. Moreover, we applied positive blood culture broths instead of a bacterial colony, when we identify microorganism caused with the infectious disease such as bacteremia and sepsis. At this time, we described the outline about the identification of microorganisms by MALDI-TOF MS, and its application to rapid diagnosis of infectious disease.
There had been no practical methods for prevention of crops from aflatoxin contamination due to lack of effective antifungal agents for aflatoxigenic fungi in fields. However, a novel biocontrol method using non-aflatoxigenic strains of Aspergillus flavus has recently become practically useful for aflatoxin contamination. In this article, usefulness and problems of new biocontrol methods for aflatoxin control are reviewed.
Aflatoxigenic fungi produce aflatoxins B1 （AFB1）, B2 （AFB2）, G1 （AFG1）, G2 （AFG2） as major aflatoxins, and some of them also produce one or a few of aflatoxins M1 （AF M1）, M2 （AFM2）, GM1 （AFGM1）, GM2 （AFGM2） as minor aflatoxins, which are 12 c-hydroxy derivatives of the major aflatoxins. Although biosynthetic pathway of the major aflatoxins has been mostly clarified, that of the minor ones has not been done. Aspertoxin is the 12c-hydroxy derivative of O-methylsterigmatocystin, which is a precursor of both AFB1 and AFG1. We recently reported that both AFM1 and AFGM1 were produced from aspertoxin in both feeding and cell-free experiments, and aspertoxin was produced from OMST through hydroxylation catalyzed by OrdA enzyme encoded by ordA gene in the aflatoxin gene cluster. On the other hand, AFM1 was not significantly produced from AFB1 in feeding experiment. These results demonstrated that the biosynthetic pathway of the minor aflatoxins in fungi is different from that of AFM1 from AFB1 in animal livers.