Full paper Synthesis of Japanese Boletus edulis ectomycorrhizae with Japanese red pine Naoki Endo a, *, Fuminori Kawamura b , Ryoko Kitahara d , Daisuke Sakuma e , Masaki Fukuda a,b , Akiyoshi Yamada a,b,c a Department of Bioscience and Food Production Science, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 8304, Minami-minowa, Nagano 399-4598, Japan b Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, 8304, Minami-minowa, Nagano 399-4598, Japan c Division of Rural Environmental and Symbiotic Science, Institute of Mountain Science, Shinshu University, 8304, Minami-minowa, Nagano 399-4598, Japan d Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan e Osaka Museum of Natural History, 1-23, Nagai-koen, Higashisumiyoshi-ku, Osaka, Osaka 546-0034, Japan article info Article history: Received 9 August 2013 Received in revised form 18 November 2013 Accepted 19 November 2013 Available online Keywords: Ectomycorrhizal morphology In vitro mycorrhization ITS phylogeny Pinus densiflora Porcini mushroom cultivation abstract Boletus edulis is a well-known ectomycorrhizal mushroom. Although cultivation has been widely attempted, no artificial fruiting has been achieved owing to difficulties associated with mycorrhizal synthesis and acclimatization in fields. We collected fifteen B. edulis basidiomata samples from locations in Japan and identified them microscopically and by phylogenetic analysis of their nuclear ribosomal internal transcribed spacer (ITS) regions. Pure culture isolates of B. edulis were established efficiently on malt extract agar medium, and one isolate, EN-63, was inoculated to axenic Pinus densiflora seedlings in vitro. Brownish ectomycorrhizal tips were observed on the pine lateral roots within four months of inoc- ulation. Ten pine seedlings that formed ectomycorrhizae were acclimatized under labo- ratory and greenhouse conditions. At four months after transplant, mycorrhizal colonization by B. edulis was observed on newly grown root tips under laboratory condi- tions, but no B. edulis ectomycorrhiza survived under greenhouse conditions. These results suggest that B. edulis ectomycorrhizae synthesized in vitro with P. densiflora requires additional steps for acclimatization to greenhouse conditions. ª 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. 1. Introduction Boletus edulis Bull. is a delicious edible mushroom known as “king bolete”, “cep”, or “porcini” (Hall et al. 1998; Boa 2004; de Roma ´ n and Boa 2004; Wang and Hall 2004; Arora 2008; A ´ gueda et al. 2008a; Dentinger et al. 2010; de la Varga et al. 2012; Feng et al. 2012). Boletus edulis belongs to the family Boletaceae in the order Boletales and phylum Basidiomycota. It is naturally and widely distributed in the Northern Hemisphere, is present in New Zealand as an exotic (Wang et al. 1995; Hall et al. 1998; Dentinger et al. 2010; Feng et al. 2012), and forms * Corresponding author. Tel.: þ81 265 77 1631; fax: þ81 265 77 1629. E-mail address: caesareae@yahoo.co.jp (N. Endo). Available online at www.sciencedirect.com journal homepage: www.elsevier.com/locate/myc mycoscience 55 (2014) 405 e416 1340-3540/$ e see front matter ª 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.myc.2013.11.008