JSM JSM 65（1）, 19 - 23（2015） Mycotoxinsins http://dx.doi.org/10.2520/myco.65.19

Review www.jstage.jst.go.jp/browse/myco Historical review of researches on yellow and mycotoxigenic fungi adherent to rice in

Masayo Kushiro1

1Food Safety Division, National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan

Keywords Abstract citreoviridin; ; “Yellow rice” is the collective name of rice grains contaminated by Peni- cyclochlorotine (islanditoxin); cillum fungi in Japan. Three kinds of yellow rice, which are caused by differ- luteoskyrin; Penicillium ent sub-species of toxigenic fungi, are known. Initially, mal-nutrition resulting from Asian poverty was regarded as the source of illness in people; however, Correspondence thanks to ongoing research, the close relationship of “Shoshin-kakke (heart- Masayo Kushiro, Food Safety attacking paralysis)”, “”, and “rice” was elucidated. Subsequently, Division, National Food Research the illness was shown to derive from “yellow rice”, which was due to grains Institute, National Agriculture and damaged by Penicillium spp. After World War II, another two kinds of yellow Food Research Organization, 2-1-12 rice, “Islandia yellow rice” and “Citrinum yellow rice”, were found, and the Kannondai, Tsukuba, Ibaraki causative toxic compounds were determined through interdisciplinary col- 305-8642, Japan. laborative studies. This article summarized the researches on toxigenic fungi E-mail: [email protected] adherent to rice and three kinds of yellow rice identified in Japan. (Received December 20, 2014, accepted January 30, 2015)

domestic rice. Introduction In 1934, the Rice Utilization Institute, a predecessor Rice is the primary staple food for Japanese. of the National Food Research Institute, was built to Although mycotoxin contamination in rice has been conduct research on the utilization and processing of reported less often than that in wheat and corn, there rice. Ichiro Miyake, Hiroshi Naito and Hiroshi Tsunoda have been occasional reports of mycotoxin contamina- were leading researchers on fungi that spoiled rice tion in stored or imported rice in Japan. grains during storage at the Rice Utilization Institute. The name “yellow rice” (ou-hen-mai; in Japanese; This review describes the classification of diseased rice “mai” being Japanese for “rice”), or rice grains affected grains and provides a historical perspective of yellow rice. and colored by , is notorious case in the history of Japanese food hygiene. However, the details of “yellow Diseased rice grains rice” are not well known. It is described that “Yellow rice disease … mainly occurred … after World War II in 1. Classification Japan. Many human deaths have been reported …” in Rice grains affected by insects or microorganisms one famous handbook on mycotoxins1). However, the are termed diseased rice grains. The systematic classifi- part of the description on human deaths is incorrect, cation of grain infected by microorganisms was origi- and actually, no human deaths occured due to nally conducted by Kingo Miyabe, Takiya Kawakami, imported yellow rice after World War II, thanks to col- Mitsutaro Shirai, I. Miyake and other researchers (sum- laborative research and preventive act of researchers marized in reference)4). Twenty-four kinds of unhealthy against the Japanese government. rice grains are known, of which 18 are molded rice The toxicity of molded rice grains in Japan was first grains spoiled by fungi3). Diseased rice grains include reported in 1891 in regard to rice affected by Aspergil- not only grains spoiled in the field but also grains lus species2). Research on diseased rice grains was spoiled after harvest. The classification summarized by started systematically from plant physiological point of Shun-ichi Udagawa and Takashi Tatsuno is shown in view. The name yellow rice refers to grains affected by Table 1 and 2, which represents fungi adherent to stand Penicillium species after harvest3). This name can be rice in the field and fungi adherent to stored rice after used not only for imported rice but also for stored harvest, respectively4). 20 KUSHIRO JSM Mycotoxins

Table 1 Rice grains spoiled in the field color from green to blue-green that covers on the sur- Name of face of the grain. Moss-rice caused by Aspergillus versi- Color of rice Causative fungi Mycotoxin diseased rice color, Versi-moss-rice, produces the mycotoxin Absidia Unknown sterigmatocystin (Table 2). Rice grains stored in some Grayish Fuke-rice Mucor Unknown Japanese storehouses at room temperature were found white Rhizopus Unknown to be contaminated with sterigmatocystin, with a peak 5) Misidentified Trichoderma level of 16.3 ppm reported in 1975 . Yellow Unknown yellow rice species 3. Penicillium species adherent to rice Acremonium Unknown Fungi of Penicillium species are frequent contami- Alternaria Unknown Brown Browny rice nants in stored rice as well as Aspergillus species Curvularia Unknown (Table 2). After long-term preservation of rice, Versi- Phoma Unknown moss-rice (A. versicolor), Yellow-spot rice (Eurotium Epicoccum Vermilion rice Unknown amstelodami) (Eurotium is a teleomorph of Aspergillus), nigrum and Citrinum yellow rice (P. citrinum) are frequently Purplish Oospora species Red rice Unknown found. red (tentative) Furthermore, fungi of Penicillium species adherent Fusarium Fusarium Trichothecenes, to stored rice are often found to produce mycotoxins ear blight species zearalenone (Table 2). Ruguro-moss-rice, Biruma-moss-rice, and Moss-rice are sometimes contaminated with rugulosin, Table 2 Rice grains spoiled after harvest penicillic acid and cyclopiazonic acid, respectively. Color of Name of Fungi responsible for yellow rice also belong to Penicil- Causative fungi Mycotoxin rice diseased rice lium species. Three kinds of yellow rice differ in the Aspergillus causative sub-species of genus Penicillium are termed White-kouji-rice Unknown Grayish candidus white “Yellow rice (P. c i t re o n i g r u m )”, “Citrinum yellow rice (P. Versi-moss-rice A. versicolor Sterigmatocystin citrinum)” and “Islandia yellow rice (P. islandicum)” Eurotium Yellow-spot rice Unknown (Table 2, in bold). The inoculation of these fungi on to rice amstelodami resulted in pale-yellow coloration (Fig. 1). The compli- Yellow rice Penicillium Citreoviridin cated chemical structures of causative mycotoxins were (Ou-hen-mai) citreonigrum elucidated through endurance of researchers (Fig. 2). Citrinum yellow Yellow rice (Citrinum P. citrinum Citrinin ou-hen-mai) Penicillium species and yellow rice Ruguro- P. r u g u l o s u m Rugulosin moss-rice 1. Shoshin-kakke (heart-attacking paralysis) and Biruma- "Yellow rice" P. aurantiogriseum Penicillic acid moss-rice The beginning of the study of mycotoxins in rice in Nicaragua- Emericella Japan can be regarded as the toxicity study of molded Sterigmatocystin browny-rice nidulans rice conducted by Junjiro Sakaki in 18912). In those Cyclopiazonic days, people who ate polished rice as a staple diet Moss-rice P. commune Brown acid tended to suffer from Shoshin-kakke (heart-attacking Islandia yellow Cyclochlorotin, paralysis) in urban area of Asia including Japan. From rice (Islandia P. islandicum luteoskyrin ethanol extracts of molded rice, Sakaki elucidated the ou-hen-mai) existence of substances that provoked similar symp- Shade-moss-rice A. restricus Unknown Dark toms to heart-attacking paralysis in experimental ani- Blacking rice Eurotium chevalieri Unknown mals. This observation led him to conclude that molded rice contained mycotoxin(s) which could cause paralysis. The study of molded rice was taken over by Miyake 2. Fuke-rice and Moss-rice et al. at Rice Utilization Institute. In 1937, they isolated a Fuke-rice and Moss-rice are empirical names of Penicillium species from yellow-colored Taiwanese rice imported into Japan6). Miyake prepared molded rice, its two types of molded rice grains. The former is a rice toxicity was analyzed by Kenji Uraguchi at The Univer- grain spoiled by fungal hyphae under humid condi- sity of Tokyo, and a causative toxic compound was ana- tions, and it appears as grayish hyphae enclosing the lyzed by Yoshimasa Hirata (later at ). grain. The causative fungi of Fuke-rice are genus The study was interrupted by the outbreak of World Absidia, Mucor, and Rhizopus, but these fungi are gen- War II and then resumed after the war. The chemical erally not mycotoxigenic (Table 1). Moss-rice is rice structure of the causative mycotoxin, named citreoviri- grains spoiled by fungal spores, which give a moss-like din, was finally elucidated in 1964 by Hirata at Nagoya Vol. 65, No. 1, 1923（2015） 21

"Islandia yellow rice" and "Citrinum yellow rice" after World War II

1. "Islandia yellow rice" World War II ended in 1945; however, exhaustion of food production in Japan forced the Japanese govern- ment to import large quantities of rice from abroad. During this period, Japan imported 1 million tons of rice, which corresponded to one-quarter of world rice Fig. 1 Rice grains inoculated with Penicillium fungi related to production. Rice was imported from all over the world. yellow rice. The Rice Utilization Institute was re-established as the Food Control Bureau Institute in 1947. In 1948, in imorted rice from , Hiroshi Tsu-

OH OH noda found a different type of diseased rice unlike the CH OCH3 HO 3 HOOC O CH3 CH3 yellow rice found previously. This molded rice induced O 9) O CH3 hobnailed liver in rats . However, the toxicity of this O O CH3 CH3 CH3 CH3 strain was unstable and there was difficulty in repro- (a) (b) ducing the effect. Subsequently, this strain was identi- fied and named P. islandicum Sopp10), and rice grains

Cl contaminated by this were called Islandia yellow OH O OH CH2OH Cl rice. OH O C NH CH C N O The occurrence of P. islandicum in Japanese rice is OH O CH C 3 H2C O rare, because the optimum growth temperature of this H3C O OH HN CH H HO species is 30 ºC, and Islandia yellow rice was found HN C CH N C CH OH O OH O occasionally in grains imported from Burma (Myan- O CH2OH mar), Thailand, and Egypt. (c) (d) Starting in 1953, a new strain was isolated from Californian rice, and Yoshito Kobayashi, K. Uraguchi, Fig. 2 Chemical structures of yellow rice toxins (a) citreoviri- din, (b) citrinin, (c) luteoskyrin, and (d) cyclochlorotine (island- Masashi Miyake, Mamoru Saito, Makoto Enomoto itoxin) formed a research team for Islandia yellow rice. In 1954, Takashi Tatsuno joined the team and the collaborative study among mycology, pharmacology, pathology, and chemistry were started. In a period of 7 months, two University (Fig. 2)7). Using pure chemical standard different types of toxic compounds were isolated from citreoviridin, Uraguchi successfully induced the symp- dried fungal material. One was a slow-acting mycotoxin toms of heart-attacking paralysis in an experimental with yellow color (originally named pigment X), and animal model8). another was a fast-acting colorless mycotoxin11),12),13), 14). Tatsuno revealed the chemical structure of pigment 2. Historical value of the research on "Yellow rice" X as anthraquinone. Before the discovery of yellow rice in Japan, other As described, the toxic components of P. islandicum mycotoxicoses caused by ergot fungus (Claviceps pur- were very unstable. A metabolite from one strain, NRRL purea) and Fusarium species were known. Both 1036, named pigment a, was investigated by Shoji Shi- occurred in the field and not after harvest. Yellow rice bata at The University of Tokyo14) was the first example of food mycotoxicosis caused by . Shibata elucidated fungi adherent to crops after harvest. Research on yel- the structure of pigment a, which was revealed to be low rice was conducted in Japan before the outbreak of the same compound as pigment X. After discussions aflatoxin mycotoxicosis in 1960 in England. The earlier between Shibata and Tatsuno, this compound was 15),16),17) research related to yellow rice in Japan is an important named luteoskyrin (Fig. 2) . event in the history of global food hygiene. As for the fast-acting colorless mycotoxin, the elu- During research of yellow rice, rice hygiene stan- cidation of chemical structure was a very painstaking dards set by the government were reinforced, and as a task because of the low amount present in fungal cul- result the incidence of Shoshin-kakke was reduced. tures compared with luteoskyrin. Finally, Shingo Considering the findings obtained from research and Marumo at Nagoya University and Tatsuno proposed 18) 19) 20) data on the disease epidemic, Shoshin-kakke should be its chemical structure independently , , . Marumo considered to be a result of mycotoxicosis and not and Tatsuno confirmed that the proposed chemical attributable to a lack of vitamin B1, as is the case for structures were identical, and this compound was other “kakke” (beriberi). named cyclochlorotine (islanditoxin) (Fig. 2)18),19),20). 22 KUSHIRO JSM Mycotoxins

2. Historical value of the research on "Islandia yellow project for cancer research funded by Ministry of rice" Health26). Research on Islandia yellow rice was conducted by In 1967, domestic rice production in Japan reached many experts of different disciplines. That both luteos- 14 million tons, and the dependence of the Japanese kyrin and cyclochlorotine (islanditoxin), show hepato- staple diet on imported rice came to an end. toxicity but with distinct toxicities, is worthy of remark However, some importation of rice still takes place, 21),22),23). The induction of hepatic cancer by luteoskyrin and in 2008, a sample of imported rice was found to be 24) was also reported by Uraguchi . tainted with aflatoxin B1, the most toxic mycotoxin. Contamination of rice with mycotoxins can still occur, 3. "Citrinum yellow rice" and monitoring efforts must continue to ensure public Citrinum yellow rice was also discovered after safety. World War II. The toxic compound was identified as citrinin (Fig. 2), which was also known as an antibiotic. The health issues caused by Citrinum yellow rice were related to the economic problems of the Japanese gov- Acknowledgements ernment and became an object of public concern. This article was written in response to the kind sug- Citrinum yellow rice was first found by Tsunoda in gestion of the Editor-in-Chief of the Japanese Society 1951 in imported rice from Thailand. In 1954, causative of Mycotoxicology, Dr. Makoto Kimura. The author is fungus was identified as P. citrinum Thom. P. citrinum grateful for sincere advice from Dr. Michihiko Saito and was detected in grains from , Vietnum, Burma Dr. Kenji Tanaka (National Food Research Institute). (Myanmer), , Spain, America, Colombia, , and also in Japanese rice. 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