TITLE	Improvement of techniques for purification of leafroll associated closterovirus from affected grapevines and the preparation of antisera for disease indexing
AUTHOR	Huey-Lin Chen Tzeng Department of Plant Pathology and Entomology, Taiwan Tobacco Research Institute, Tsao-Hu, Taichung 412, Taiwan Moh-Jih Chen Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan Dean Der-Syh Tzeng Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan
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ABSTRACT	An improved technique was developed for the purification of leafroll associated closterovirus (GLRaV) and successfully applied for the preparation of polyclonal and monoclonal antisera for the virus detection. The technique improvement mainly involves the use of a Chinese herb grinder for the homogenization of diseased sample. It requires very limited labor, is repeatable in regard to the virus yield, and can process considerable quantities within a short period. By this sample homogenization technique and a method modified from one in the available literature, GLRaV was successfully purified from diseased Kyoho, Black Queen, and Golden Mutscate grapevines cultivated in Taiwan. The electron microscopic examination of the obtained virus preparations revealed that the yield and purity were comparable or better than that reported by previous workers. With the obtained virus samples, polyclonal and monoclonal antisera were prepared. The results obtained from direct ELISA (enzyme linked immunosorbent assay) and indirect ELISA revealed that the prepared antisera detected mainly type III and type IV GLRaV. With the immunological method established, a disease survey was conducted between the years 1987 and 1994. A total of 204 grapevine cultivars or breeding lines were examined, and approximately 52% of them were found affected by GLRaV. Among the surveyed grapevines, about 30% were affected by Type III virus and another 31% by Type IV. A complexed infection of Types III and IV appeared to be common among Kyoho grapevines. The results also indicated the natural spread of GLRaV in tested field and suggested the need of a strict sanitation program for important breeding stock conservation.
KEYWORD	Antiserum preparation; Chinese herb grinder; Disease indexing; Grapevine leafroll disease; Virus purification;
ARTICLE INFO	Botanical Bulletin of Academia Sinica, Volume 40 Number 4 October 1999, page 295-304, 10 pages
PUBLISHER	Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China