Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan Pierce’s Disease of Grapevines in Taiwan: Isolation, Cultivation and Pathogenicity of Xylella fastidiosa Chiou-Chu Su 1 , Chung Jan Chang 2,3 , Che-Ming Chang 1 , Hsien-Tzung Shih 4 , Kuo-Ching Tzeng 3 , Fuh-Jyh Jan 3 , Chin-Wen Kao 1 and Wen-Ling Deng 3 Authors’ addresses: 1 Division of Pesticide Application, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Wufeng, Taichung 41358, Taiwan; 2 University of Georgia, Griffin, GA 30223, USA; 3 Department of Plant Pathology, National Chung Hsing University, Taichung 40227, Taiwan; 4 Applied Zoology Division, Taiwan Agricultural Research Institute, Wufeng, Taichung 41362, Taiwan (correspondence to W.-L. Deng. E-mail: wdeng@nchu.edu.tw) Received October 4, 2012; accepted December 22, 2012 Keywords: Xylella fastidiosa, grapevine, leaf scorch disease, 16S rRNA, 16S-23S ITS, Taiwan Abstract Characteristic symptoms of Pierce’s disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investi- gated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch’s postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Mus- cat. The inoculated plants developed typical leaf- scorching symptoms, and similar disease severity devel- oped in the three cultivars from which the bacterium was readily re-isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S-23S rRNA internal transcribed spacer region (16S-23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from dif- ferent hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other. Introduction Xylella fastdiosa is a gram-negative, non-flagellate, nutritionally fastidious bacterium that occurs only in xylem tissues and requires specific and enriched media for in vitro growth (Wells et al. 1987). X. fastdiosa causes many economically important diseases, includ- ing Pierce’s diseases (PD) of grapevine (Davis et al. 1978), citrus variegated chlorosis (Chang et al. 1993; Hartung et al. 1994) and leaf scorch diseases of pear (Leu and Su 1993), elm (Hearon et al. 1980), blueberry (Chang et al. 2009), almond (Mircetich et al. 1976), oak (Chang and Walker 1988), plum (Raju et al. 1982), peach (Wells et al. 1983), mulberry (Kostka et al. 1986) and oleander (Purcell et al. 1999). Pierce’s disease of grapevines has been reported from countries in the Americas, including Mexico and the Unites States in North America, Costa Rica and Venezuela in Central America and Caribbean, Argen- tina and Peru in South America. In Europe Kosovo, former Yugoslavia, is the only country in which PD incidence has been reported (Berisha et al. 1998). PD has been a limiting factor for the establishment of the wine industry in the south-eastern United States along the Gulf of Mexico, and the emergence of glassy- winged sharpshooter (GWSS), Homalodisca vitripennis (formerly Homalodisca coagulata), in California, has alarmingly increased the PD incidence. From 1994 to 2000, the disease destroyed more than 1000 acres of vineyards in northern California, causing losses of $30 million US dollars. There has been no report of PD in Asian Continent, except the report of a suspected PD- like disorder in Shaanxi province, China, on cv. Red Globe grapevines that were originally imported from California (Chu 2001). In Taiwan, grape cultivation is a remunerative agri- business for the highly popular grape berries among local consumers in addition to the added values with the processed products. The annual production of grapes was 102 831 metric tons in 2010, and 99.5% was produced in Taichung City, Miaoli, Changhua and Nantou Counties in central Taiwan (AG Statistics Yearbook 2010, Agriculture and Food Agency, COA, Executive Yuan, Taiwan). Popular cultivars include J Phytopathol 161:389–396 (2013) doi: 10.1111/jph.12075 © 2013 Blackwell Verlag GmbH