Risk analysis and development of a rapid method for identifying four species of Byssochlamys Kouichi Hosoya a , Motokazu Nakayama a , Tetsuhiro Matsuzawa b , Yumi Imanishi b , Jun Hitomi a , Takashi Yaguchi b, * a Global R&D-Safety Science, Kao Corporation, 2606 Akabane, Ichikai-machi, Haga-Gun, Tochigi 321-3497, Japan b Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan article info Article history: Received 31 August 2011 Received in revised form 4 January 2012 Accepted 10 January 2012 Keywords: Byssochlamys Patulin Heat-resistant fungi PCR Identification to species level abstract Species of the genus Byssochlamys, heat-resistant fungi, cause the spoilage and poisoning of heat- processed acidic foods due to the formation of heat-resistant ascospores and the biosynthesis of patu- lin. Here we evaluated the heat resistance and patulin production of four species of Byssochlamys. Byssochlamys fulva was significantly more heat-resistant than the other species, and Byssochlamys lagunculariae and Byssochlamys nivea synthesized patulin. This is the first report of patulin production by B. lagunculariae. These findings indicate that the four species pose different health risks in food. Thus, we concluded that a method to identify the species of Byssochlamys is necessary. Identification at the species level based on morphology alone is difficult. We developed species-specific primers to identify B. nivea, B. fulva, B. lagunculariae, and Byssochlamys zollerniae using polymerase chain reaction (PCR) to amplify the b-tubulin gene. PCR using the primer sets designed for B. nivea (B. nivea1F/1R) amplified PCR products specific for B. nivea. Similarly, PCR using the B.fulva1F/1R, B.lag1F/1R and B.zol3F/R primer sets produced PCR products specific for B. fulva, B. lagunculariae, and B. zollerniae, respectively. PCR using these four primer sets did not detect other fungi involved in food spoilage and environmental contamination. This identification method is rapid and simple with extremely high specificity. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction In general, fungi have low heat resistance and can be pasteurized in acidic fruit juices. Heat-resistant fungi form ascospores, however, that allow them to survive at temperatures of up to 75  C for 30 min (Samson, Hoekstra, Lund, Filtenborg, & Frisvad, 2004). Of the heat- resistant fungi, Byssochlamys spp. are widely distributed in soil throughout the world, and through the contamination of agricultural crops, food products contaminated with Byssochlamys spp. reach the market (Warcup & Baker, 1963). Byssochlamys spp. have the most heat-resistant ascospores among the heat-resistant genera (Bayne & Michener,1979; Kotzekidou,1997) and are responsible for spoilage of strawberry, pineapple, passion fruit, mango, grape, and citrus fruit juices, as well as other acidic beverages and dairy products (Beuchat, 1998; Beuchat & Rice,1979; Dijksterhuis, 2007; Hull,1939; Put,1964; Samson et al., 2004; Tournas, 1994). In addition, because Byssochl- amys spp. are prolific under low oxygen conditions, the resulting production of pectinolytic enzymes, such as polygalacturonase, which alters food characteristics, and patulin, which is an important carcinogenic mycotoxin (Bonerba, Ceci, Conte, & Tantillo, 2010; Puel, Tadrist, Delaforge, Oswald, & Lebrihi, 2007; Tournas, 1994), can reduce the quality of processed fruit food products, making them the most harmful organisms in acidic food products. Control of Byssochlamys spp. by heat pasteurization is difficult, and it is thus important to prevent contamination through moni- toring of raw materials and the manufacturing environment. The most common identification method relies on morphologic examination. The high degree of similarity among these fungi (Murdock & Hatcher, 1978; Put, 1964), however, is problematic for the rapidity and general versatility of this testing. As a first step, we developed a genus-level method for identification using poly- merase chain reaction (PCR). The Byssochlamys genus comprises six species: Byssochlamys nivea, Byssochlamys fulva, Byssochlamys zollerniae, Byssochlamys spectabilis/Paecilomyces variotii, Byssochlamys lagunculariae, and Byssochlamys verrucosa (Nakayama et al., 2010; Ueda, Kawara, Yaguchi, & Udagawa, 2010). Among them, B. nivea, B. fulva, B. lagunculariae and B. spectabilis are causative agents of food spoilage (Nakayama et al., 2010; Put, 1964; Ueda et al., 2010) B. verrucosa differs phylogenetically from the other Byssochlamys * Corresponding author. Tel.: þ81 43 226 2790; fax: þ81 43 226 2486. E-mail address: t-yaguchi@faculty.chiba-u.jp (T. Yaguchi). Contents lists available at SciVerse ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.01.024 Food Control 26 (2012) 169e173