International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1543–1549 DOI : 10.1099/ijs.0.01980-0 ‘ Candidatus Phytoplasma castaneae ’, a novel phytoplasma taxon associated with chestnut witches’ broom disease 1 Laboratory of Plant Pathology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113- 8657, Japan 2 Laboratory of Bioresource Technology, The University of Tokyo, 202 Frontier Bioscience Building, Kashiwa, Chiba 277-8562, Japan 3 Laboratory of Plant Pathology, Kyungpook National University, Taegu 702-701, Korea Hee-Young Jung, 1 Toshimi Sawayanagi, 2 Shigeyuki Kakizawa, 2 Hisashi Nishigawa, 2 Shin-ichi Miyata, 2 Kenro Oshima, 2 Masashi Ugaki, 2 Joon-Tak Lee, 3 Tadaaki Hibi 1 and Shigetou Namba 2 Author for correspondence : Shigetou Namba. Tel : 81 471 36 3700. Fax: 81 471 36 3701. e-mail : snambaims.u-tokyo.ac.jp In Korea, Japanese chestnut trees (Castanea crenata Sieb. and Zucc.) showing symptoms indicative of witches’ broom disease, including abnormally small leaves and yellowing of young leaves, were examined. Since the symptoms were suggestive of a phytoplasma infection, tissues were assayed for phytoplasmas by PCR analysis using a pair of universal primers that amplify a 14-kbp phytoplasma 16S rDNA fragment. The phytoplasma-specific fragment was amplified from diseased plants, but not from healthy plants, indicating that a phytoplasma was the causal agent of the chestnut witches’ broom (CnWB) disease. The phylogenetic relationship of the CnWB phytoplasma to other phytoplasmas was examined by sequence analysis of the 16S rDNA. A phylogenetic analysis of 16S rDNA sequences of the phytoplasmas placed the CnWB phytoplasma within a distinct subgroup in the phytoplasma clade of the class Mollicutes. The phylogenetic tree indicated that the CnWB phytoplasma is related most closely to coconut phytoplasmas and suggested that they share a common ancestor. The unique properties of the CnWB phytoplasma sequences clearly establish that it represents a novel taxon, ‘ Candidatus Phytoplasma castaneae ’. Keywords : ‘ Candidatus Phytoplasma castaneae ’ INTRODUCTION Phytoplasmas are prokaryotes that lack a cell wall and are the causal agents of numerous plant diseases (McCoy et al., 1989 ; Kirkpatrick, 1992). Phytoplasmas inhabit sieve elements in the phloem of plants and are transmitted between plants by phloem-feeding insects. Phytoplasmas are small enough to pass through bacteriological filters and, like mycoplasmas, are re- sistant to antibiotics that interfere with cell-wall formation. Chestnut trees (Castanea spp.) are grown widely in the temperate zone of the northern hemi- sphere as commercially important vegetable crops. The chestnut phytoplasma disease was first described tentatively as a graft-transmissible viral disease in ................................................................................................................................................. Abbreviations : CLL, chestnut little leaf ; CnWB, chestnut witches’ broom. The EMBL/DDBJ/GenBank accession number for the 16S rDNA sequence of ‘ Candidatus Phytoplasma castaneae ’ reported in this paper is AB054986. Japanese chestnut, Castanea crenata Sieb. and Zucc., in Japan, and was named chestnut yellows disease (Shimada & Kouda, 1954). However, it was later shown by transmission electron microscopy that a phytoplasma was associated with the disease (Okuda et al., 1974). In Korea in 1993, chestnut trees showing the typical symptoms of small, yellow leaves were examined. Based on electron and fluorescence micro- scope analysis, a phytoplasma was implicated as the aetiological agent of the disease (Han et al., 1997). It was named chestnut little leaf (CLL) disease, reflecting the most obvious symptom. In Italy, European chest- nut trees (Castanea sativa Mill) showing small, yellowing leaves have also been found in chestnut orchards, but it is still not known whether the causal disease was associated with phytoplasmas (Mittem- pergher & Sfalanga, 1998). In Korea, Japanese chestnut trees showing the typical witches’ broom symptoms, suggestive of phytoplasma 01980  2002 IUMS Printed in Great Britain 1543