Caracterização parcial de um Tymovirus isolado de tomateiros

Um Tymovirus isolado de sementes de tomateiro (Solanum lycopersicum) 'Paronset' com sintomas de necrose, denominado TyToRS08, foi caracterizado quanto ao círculo de hospedeiras, propriedades físico-químicas, sorológicas e moleculares. O vírus foi transmitido mecanicamente a partir de folhas, frutos e sementes infectadas. A gama de hospedeiras incluiu espécies de Amaranthaceae, Chenopodiaceae e Solanaceae, induzindo sintomas local e sistêmicos na maior parte das espécies, exceto para Nicotiana sylvestris, N. tabacum 'Samsun', 'White Burley' e 'Xanthi' que não manifestaram sintomas. Em tomateiros 'Alambra', 'Thomas', 'Diva', 'Romana' e 'Graziela', o vírus induziu sintomas de mosaico e necrose branca. A presença do vírus foi detectada, nas sementes infectadas de todas as cultivares avaliadas, porém não se notou em plântulas após a germinação destas sementes. Foi obtida uma preparação purificada com concentração de 1,04 mg mL-1, empregada na produção de um antissoro policlonal que reagiu em PTA-ELISA com um título de 1/32768. Utilizou-se um par de oligonucleotídeos degenerados, desenhados para anelar na ORF 1 de espécies de Tymovirus, que permitiu a amplificação por RT-PCR de fragmentos com cerca de 700 pb. Após o alinhamento e a análise das sequências, verificou-se que o isolado TyToRS08 possuía valores abaixo de 70% de identidade com as espécies de Tymovirus, indicando que pode se tratar de uma espécie distinta das descritas no gênero, com potencial epidemiológico, pela sua estabilidade e disseminação por sementes de tomate.

Evaluation the efficiency of different techniques for extraction and purification of Tomato yellow leaf curl virus (TYLCV)

This study was conducted to evaluate the efficacy of different techniques for extraction and purification of Tomato yellow leaf curl virus (TYLCV). An isolate of the virus free of possible contamination with other viruses infecting the same host and transmitted by the same vector Bemisia tabaci Genn. was obtained. This was realized by indicator plants and incubation period in the vector. Results obtained revealed that the virus infect Nicotiana glutinosa without visible symptoms, while Nicotiana tabaccum var. White Burley was not susceptible to the virus. The incubation period of the virus in the vector was found to be 21 hrs. These results indicate that the virus is TYLCV. Results showed that Butanol was more effective in clarification the sap and eliminate of plant proteins and chlorophyll. The use of citrate buffer at pH 8 amended with reducing agents and EDTA to prevent the oxidation of phenolic compound was found to be suitable in maintaining the biological activity of the virus during extraction. The quantity of the virus obtained was 3.05 mg/100 gm leaves with absorption ratio of 1.4 at 260/280 nm which represent standard value for TYLCV.

First Report of Tobacco vein banding mosaic virus in China (Xian, Shaanxi Province) in Datura stramonium and Tobacco

Tobacco vein banding mosaic virus (TVBMV) has been reported in Taiwan (1), North America (Tennessee) (2), and Japan (3) and induces a severe disease of tobacco. During surveys on viruses of vegetables in China, TVBMV was isolated from a Datura stramonium weed plant in July 1998 in Shaanxi Province. It showed severe mosaic with blistering of the leaves. The plant was also infected by Cucumber mosaic virus (CMV). When sap from D. stramonium was frozen, thawed, and mechanically inoculated, only TVBMV was recovered. The 3′-end of the viral genome was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using primers derived from the potyviridae primers (4) and cloned in pBlueScript. The sequence of 1,630 bp (GenBank AF274315) was determined on both DNA strands and found to have approximately 94% homology with other TVBMV sequences (L 28816 from Tennessee, X77637 from Taiwan, and AB020524 from Japan). The host range of the Chinese isolate was similar to that reported for the U.S. isolate. D. stramonium, Nicotiana benthamiana, N. clevelandii, N. glutinosa, N. tabacum Samsun, White Burley type and Xanthi, Lycopersicon esculentum cv. Marmande, and Petunia hybrida were systemically infected. A local infection developed in N. rustica, Chenopodium amaranticolor, C. quinoa, and Ocimum basilicum. The Chinese isolate did not infect Capsicum annuum cv. Quadrato d'Asti, Solanum melongena, or several Cucurbitaceae and Leguminosae species. Myzus persicae transmitted the Chinese TVBMV in a non-persistent mode from both D. stramonium and tobacco to the same plants and to tomato. No seed transmission occurred in experimentally infected D. stramonium (20 seedlings), tobacco White Burley type (200 seedlings), and tomato cv. Marmande (100 seedlings). The virus was found in the roots of D. stramonium and tobacco. Since the virus was not seed-transmissible, overwintering rootstocks may provide sites for winter survival of the virus. An antiserum was produced against the virus and an enzyme-linked immunosorbent assay survey was carried out in solanaceous crops including D. stramonium collected in July 1999 in Shaanxi, Shanxi, Henan, and Hebei provinces and Beijing surroundings. TVBMV was found only in the same field as in 1998 in four D. stramonium plants in association with CMV and in a tobacco plant 200 m from D. stramonium. TVBMV was not found in the closest tomato crops, where infection of CMV was severe. This is the first report of TVBMV in China, and Xian is the most northern location in which this virus has been found. References: (1) J. K. Chiang et al. Bull. Tobacco Res. Inst. 32:39, 1990. (2) B. B. Reddick et al. Plant Dis. 76:856, 1992. (3) H. Tochihara. Rev. Plant Prot. Res. 13:122, 1980. (4) A. Gibbs and A. Mackenzie. J. Virol. Meth. 63:9, 1997.