Inoculation, Purification and Serology of Tobacco Leaf Curl Virus

Tobacco leaf curl isolates were collected from tobacco fields throughout Japan. Diseased plants from southern Japan showed severe symptoms, whereas plants from northeastern Japan showed mild symptoms. Results of graft inoculation experiments in the green house showed that the difference in symptoms was dependent on environmental conditions, not on virus strains. Isolates from all areas developed similar symptoms under green house conditions. Sixteen new host species belonging to genus Nicotiana were found. These included N. knightiana, N. pauciflora and N. bigelovii. Several methods were tested in an attempt to transmit tobacco leaf curl virus (LCV) mechanically, but with no success.LCV was partially purified from the sap of infected tobacco leaves by butanol clarification and differential centrifugation. Purified preparations exhibited a single peak during analytical ultracentrifugation, and showed ultraviolet absorption maxima at 260mμ and minima at 238mμ. Negative stained virus preparations showed spherical particles approximately 30mμ in diameter under an electron microscope.Anti-LCV serum, titre 1/64, was obtained from rabbits injected with the purified virus preparations. Results of precipitin tests, agar diffusion tests and absorption tests between the anti-LCV serum and homologous and heterologous antigens, confirmed that the antiserum contained LCV specific antibodies. The usefulness of serological technique with non-sap transmissible virus is discussed.