First Report of Chrysanthemum stem necrosis virus on Chrysanthemums in Japan.

The chrysanthemum (Dendranthema grandiflorum), whose planted area comprises more than 6,000 ha in Japan, is one of the most important ornamental cut flower crops. In August 2006, necrotic streaks on stems, chlorotic and necrotic spots and rings on leaves, and leaf distortions were observed on chrysanthemum cvs. Jimba and Seinotama, with a disease incidence of more than 70% (approximately 30,000 plants), which represents approximately 1,000 m² of greenhouses of one grower in Hiroshima Prefecture, western Japan. Symptoms were similar to those caused by Tomato spotted wilt virus (TSWV) (genus Tospovirus, family Bunyaviridae). Frankliniella occidentalis was the major thrips species observed on symptomatic plants, followed by F. intonsa. Tospovirus-like spherical particles that were 80 to 100 nm in diameter were found in the infected leaves. After mechanical inoculation, a single lesion isolate reproduced the original symptoms observed in nature on healthy chrysanthemum plants (cv. Jimba). As determined by mechanical inoculation, host range and symptomatology of the isolate were similar to those described previously for Chrysanthemum stem necrosis virus (CSNV), including necrotic spots on Petunia hybrida (1). The isolate caused stunting, severe necrotic lesions on stems, necrotic spots, rings, and vein necrosis on systemically infected leaves of Lycopersicon esculentum (cv. House-momotaro). This virus reacted strongly with CSNV antiserum (DSMZ, Braunschweig, Germany) by indirect dot immuno-binding assay, and cross-reacted weakly with a monoclonal antibody to N protein of TSWV (3) using double-antibody sandwich-ELISA. Reverse transcription (RT)-PCR was conducted to verify virus infection. No amplification was observed from extracts of symptomatic plants (n = 10) by multiplex RT-PCR using TSWV and Impatiens necrotic spot virus specific primer sets (4), indicating that the diseased chrysanthemums were not doubly infected with these viruses. However, a DNA fragment of approximately 450 bp was amplified in samples by RT-PCR using tospovirus universal primers, BR60/65 (2). The nucleotide sequence of the amplified fragment had 98.1% identity with the corresponding region of the CSNV nucleocapsid protein gene (GenBank Accession No. AF067068). The above results indicate that the virus associated with a stem necrosis disease of chrysanthemums in Hiroshima is an isolate of CSNV. To our knowledge, this is the first report of CSNV in Japan. References: (1) I. C. Bezerra et al. Phytopathology 89:823, 1999. (2) M. Eiras et al. Fitopatol. Bras. 26:170, 2001. (3) S. Tsuda et al. Ann. Phytopathol. Soc. Jpn. 60:216, 1994. (4) H. Uga and S. Tsuda. Phytopathology 95:166, 2005.