JSM Mycotoxins

Part I (Papers in English)

Research Papers

Effects of volatile compounds in apple juices on fungal growth and patulin production of Penicillium expansum

Tomoyasu TAGUCHI, Atsushi ISHIHARA, Hiromitsu NAKAJIMA

2014 Volume 64 Issue 1 Pages 1-14
Published: January 31, 2014
Released on J-STAGE: June 21, 2014

DOIhttps://doi.org/10.2520/myco.64.1

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Penicillium expansum was cultured in apple juice media prepared from six different commercial apple juices. The patulin production was profoundly affected by the differences in apple juices, whereas fungal growth was generally not. The maximum concentration of patulin was 7.3-fold of that in the media containing minimum concentration of patulin. The six apple juices were concentrated by evaporation and reconstructed to the original volumes by adding Milli-Q water. P. expansum was cultured in the media prepared from reconstructed apple juices, and the patulin concentration and fungal growth were determined. Evaporation of apple juice tended to decrease patulin production and to increase fungal growth, suggesting that the volatile compounds promote patulin production and inhibit fungal growth. The volatile compounds in the apple juice were then analyzed by GC-MS. The 13 compounds of which concentrations were largely decreased by evaporation were selected for evaluation of their stimulation of patulin production. Seven out of the 13 compounds, 2-methylpropyl acetate, ethyl butyrate, ethyl 2-methylbutanoate, 3-methyl-1-butanol, hexyl acetate, 1-hexanol, and 2-methylbutanoic acid, increased the patulin production of P. expansum concentration-dependently; 2-methylbutanoic acid and its ethyl ester were highly effective. Our results indicated that the composition of volatile compounds in apple juice media largely affects the patulin production and the growth of P. expansum.

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Determination of aflatoxin M₁ in powdered formula: an inter-laboratory study and the surveillance in Japan

Hisako SAKUMA, Yoshiko SUGITA-KONISHI, Toshitsugu TANAKA, Toshihiro NA ...

2014 Volume 64 Issue 1 Pages 15-21
Published: January 31, 2014
Released on J-STAGE: June 21, 2014

DOIhttps://doi.org/10.2520/myco.64.15

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A method to determine aflatoxin M₁ (AFM₁) levels by using an immunoaffinity column-based clean-up procedure and HPLC with fluorescence detection was validated by an inter-laboratory study among ten laboratories in Japan. Using the validated method, we surveyed AFM₁ contamination in powdered formula. Samples for validation included a blank, three levels (blind pairs) of AFM₁ spiked into liquid milk, naturally contaminated liquid milk, and naturally contaminated powdered formula. All samples were frozen and sent to the ten participating laboratories. For the liquid milk spiked at 1.0, 0.5, and 0.05 μg/kg levels, recoveries were 89.9, 91.6, and 88.2%, respectively. The repeatability relative standard deviation (RSD_r) and reproducibility relative standard deviation (RSD_R) were less than 7.4 and 8.1%, respectively. The recovery, RSD_r, and RSD_R of the powdered formula were 94.5, 8.9, and 11.9%, respectively. The RSDr and RSDR of the naturally contaminated milk were 13.3 and 20.9%, respectively. The Horwitz ratio (HorRat) values of all six samples were less than 1.0. For surveillance, 108 commercial powdered formulae were obtained in Japan. The average value of AFM₁ in the powdered formulae was 0.002 μg/L, as ready-for-infant liquid milk (14 g powdered formula in 100 mL water). The highest contamination was 0.025 μg/L.

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Technical Note

Extraction of a Fusarium mycotoxin zearalenone in edible oils

Yazhi ZHENG, Hiroyuki NAKAGAWA, Yuki SAGO, Hitoshi NAGASHIMA, Hiroshi ...

2014 Volume 64 Issue 1 Pages 23-27
Published: January 31, 2014
Released on J-STAGE: June 21, 2014

DOIhttps://doi.org/10.2520/myco.64.23

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A simple, fast, accurate analytical method of Fusarium mycotoxin zearalenone (ZEA) from edible oils was developed by analyte-extension of the method for aflatoxins in oils. Oil samples were extracted with a mixture of acetonitrile and water (9 + 1, v/v) followed by the clean-up with a multifunctional column. ZEA content was determined by high-performance liquid chromatography with fluorescence detector (HPLC-FLD). The method was in-house validated for two matrices, blended oil and olive oil. The recovery rates were between 100.4% and 105.2 %, while relative standard deviations (RSDs) were between 0.9% and 2.3%.

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Part II (Papers in Japanese)

Proceedings of the 73th meeting

“Special lecture”

Stealth infection strategy in fungal plant pathogens – can we break fungal stealth?

Marie NISHIMURA

2014 Volume 64 Issue 1 Pages 37-43
Published: January 31, 2014
Released on J-STAGE: June 21, 2014

DOIhttps://doi.org/10.2520/myco.64.37

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Plants have immunity which is activated against broad range of microbes upon recognition of ‘non-self’ molecules called PAMPs （pathogen-associate molecular patterns） such as flagellin on bacterial flagella and chitin oligomer derived from fungal cell walls. However, pathogens have developed mechanisms to overcome this host immunity for successful infection. We have studied immune evasion mechanisms in fungal plant pathogens using an ascomycete rice pathogens Magnaporthe oryzae as a model. We have revealed that M. oryzae masked cell wall PAMPs with α-1,3-glucan, a non-degradable polysaccharide in plants. Indeed, M. oryzae mutant lacking α-1,3-glucan rapidly activated host immunity. Interestingly, ascomycete Cochliobolus miyabeanus and basidiomycete Rhizoctinia solani concealed cell wall PAMPs with α-1,3-glucan during rice infection. In supporting of this finding, transgenic rice plants secreting a bacterial α-1,3-glucanase became resistant to these fungal pathogens. Our finding suggested that a wide range of fungal pathogens use α-1,3-glucan for evading host immunity. Thus, α-1,3-glucan may be a efficient target to controlling various fungal diseases. Use of α-1,3-glucanase-producing bacteria and α-1,3-glucanse will have potential applications in sustainable crop protection techniques.

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“Topics”

Mechanisms of transcriptional activation of the cellulase genes in filamentous fungi

Emi KUNITAKE, Shuji TANI, Jun-ichi SUMITANI, Takashi KAWAGUCHI

2014 Volume 64 Issue 1 Pages 45-54
Published: January 31, 2014
Released on J-STAGE: June 21, 2014

DOIhttps://doi.org/10.2520/myco.64.45

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To get a deeper insight into cellulase gene regulation mechanisms in Aspergillus aculeatus, we screened cellulose induction deficient mutants from the T-DNA insertion mutant library and identified a novel Zn（II）₂Cys₆ transcription factor, which was designated as the cellobiose response regulator （ClbR）. Functional analysis of clbR by gene disruption and overexpression revealed that ClbR regulates the cellulose and cellobiose-responsive induction of the cellulase and xylanase genes in A. aculeatus. Furthermore, we demonstrated the genetic interaction between ClbR and its paralog on the regulation of manR gene which is recently identified as a pathway specific transcriptional activator.

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“Fusarium toxin today”

State of the art in the analyisis of Fusarium toxins by liquid chromatography-mass spectrometry (LC-MS)

Hiroyuki NAKAGAWA

2014 Volume 64 Issue 1 Pages 55-62
Published: January 31, 2014
Released on J-STAGE: June 21, 2014

DOIhttps://doi.org/10.2520/myco.64.55

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A simultaneous and multiple determination method for the major five Fusarium toxins (deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin, and zearalenone) contaminating wheat and barley was developed with liquid chromatography tandem-mass spectrometry (LC-MS/MS), and a harmonized collaborative validation of the method was conducted by the participants of 12 laboratories. Although there are many reports on the simultaneous and multiple determination of Fusarium toxins by instrumental analysis, only a few of them are developed to the harmonized collaborative validation. The variance in the extraction efficiency and the matrix effects on the ionization were occasionally innegligible with the mycotoxin analysis by LC-MS/MS, however, the employment of internal standards with the present method apparently contributed to compensate for these effects. This is the first report of a simultaneous and multiple determination method for both type A and B trichothecenes along with ZEA by LC-MS/MS that was validated through the harmonized collaborative validation. On the other hand, the accurate mass and high-resolution measurement was carried out with LC-MS (LC-Orbitrap MS) for the screening of new masked mycotoxins. Consequently, monoglucoside derivatives of type B trichothecenes (nivalenol and fusarenon-X) in wheat grains and those of type A trichothecenes (T-2 toxin, HT-2 toxin, neosolaniol, diacetoxyscirpenol, and monoacetoxyscirpenol) in corn powder sample were detected, respectively. Di-glucoside derivatives were also found for T-2 toxin and HT-2 toxin. Our findings indicate that the presence of masked mycotoxins is not limited to some specific mycotoxins such as deoxynivalenol and zearalenone, but likely with the other Fusarium toxins.

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Occurrence of deoxynivalenol, T-2 toxin, HT-2 toxin and zearalenone in retail foods in Japan

Tomoya YOSHINARI

2014 Volume 64 Issue 1 Pages 63-68
Published: January 31, 2014
Released on J-STAGE: June 21, 2014

DOIhttps://doi.org/10.2520/myco.64.63

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Fusarium toxins are a group of mycotoxins produced by many kinds of Fusarium species, and frequently detected in field crops such as wheat, barley, maize and corn. They cause mycotoxicosis, a serious health hazard to humans and domestic animals. Therefore, collecting the information about fusarium toxin contamination in daily foods is crucial. Occurrence data of deoxynivalenol, a major fusarium toxin, have been collected in worldwide, but that of other toxins is limited. In this study, we examined foods from Japanese retail shops for contamination with four kinds of fusarium toxins, deoxynivalenol, T-2 toxin, HT-2 toxin and zearalenone.

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Basic research of the regulation mechanisms of trichothecene production for reduction of the mycotoxin contamination

Kazuyuki MAEDA, Yuichi NAKAJIMA, Hinayo ICHIKAWA, Yoshiyuki KITOU, Tak ...

2014 Volume 64 Issue 1 Pages 69-74
Published: January 31, 2014
Released on J-STAGE: June 21, 2014

DOIhttps://doi.org/10.2520/myco.64.69

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Novel bioactive substances that regulate trichothecene production are in great demand both as tools for basic research and as control agents for reduction of mycotoxin contamination. Using compounds from the RIKEN Natural Product Depository (NPDepo) library, we are now screening for activators and inhibitors of trichothecene production. In this symposium, we describe the outlines of our recent progress on the regulation mechanisms of trichothecene production.

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Minireviews

Elucidation of indole-diterpene mycotoxin biosynthetic pathways and identification of terpendole E as a key biosynthetic intermediate

Takayuki MOTOYAMA, Masashi UEKI, Hiroyuki OSADA

2014 Volume 64 Issue 1 Pages 75-86
Published: January 31, 2014
Released on J-STAGE: June 21, 2014

DOIhttps://doi.org/10.2520/myco.64.75

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Indole-diterpenes are a group of compounds that contain mycotoxins including paxilline and lolitrem B. Recently, similarity of indole-diterpene biosynthetic pathways has been revealed by isolating and analyzing indole-diterpene biosynthetic gene clusters. We found an indole-diterpene compound terpendole E as the first natural product inhibitor of kinesin Eg5 by screening of cell cycle inhibitors. We found that terpendole E is a key biosynthetic intermediate of indole-diterpens by isolating and analyzing the terpendole E biosynthetic gene cluster.

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Application of somatic homologous recombination induced by chemical stress in the genome of filamentous fungus to a biological marker of mycotoxins

Takayuki ARAZOE, Shuichi OHSATO, Makoto KIMURA, Tsutomu ARIE, Shigeru ...

2014 Volume 64 Issue 1 Pages 87-94
Published: January 31, 2014
Released on J-STAGE: June 21, 2014

DOIhttps://doi.org/10.2520/myco.64.87

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It has been suggested that somatic homologous recombination is one of the important mechanisms of genetic variations in asexual life cycle of filamentous fungi. Recently, a simple detection/selection system of somatic homologous recombination was constructed by using nonfunctional fluorescence antibiotic markers in the rice blast fungus Pyricularia oryzae. Here we have summarized the detection systems of somatic homologous recombination and the current advancements of genome responses for unsuitable conditions in eukaryotes. We focused on somatic homologous recombination events induced by chemical stress treatment, and discussed the application of this mechanism to a biological marker of several mycotoxins.

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Implication of sex-determing factors for gender disparity in liver cancer

Shigekazu MURAKAMI, Fumio TASHIRO

2014 Volume 64 Issue 1 Pages 95-103
Published: January 31, 2014
Released on J-STAGE: June 21, 2014

DOIhttps://doi.org/10.2520/myco.64.95

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Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men. Even though the causative factors for HCC are well known to be hepatitis B/C viruses, aflatoxin B₁ (AFB₁) and abuse of alcohol, our knowledge about the processes of HCC development is very little due to their complexity. By this reason, molecular -targeted drugs for HCC is currently very limited. In this review, we described that based on the recent reportssex-determining factors including Sry (sex determining region Y) and sex hormones play a crucial role in onset of male HCC and its conversion into malignant tumor via cancer stem cell (CSC). These findings may confer the useful information for development of promising chemoprevention and anti-cancer drugs against male HCC.

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