1.11.64
MOLECULAR CHARACTERIZATON OF TBLCV AND TYLCV OCCURRING IN JAPAN

M ONUKI, J SAKAI and K HANADA

Kyushu National Agricultural Experiment Station, Nishigoshi, Kumamoto 861 -1192, Japan

Background and objectives
Tobacco leaf curl virus (TBLCV) was known as the only geminivirus infecting solanaceous plants in Japan. Recently, yellow leaf curl disease of tomato caused by tomato yellow leaf curl virus (TYLCV) is prevalent in various parts of Japan. Therefore we discuss the molecular characterization of several isolates of T BLCV and TYLCV.

Materials and methods
Geminiviruses were derived from Eupatorium makinoi (TbLCV-Eu), honeysuckle (TbLCV-Hs), and tomatoes (TYLCV-Nagasaki, -Ng; TYLCV-Shizuoka, -Sz). Total nucleic acid was extracted from infected and healthy plants. PCR amplification was carried out using universal primers [1] and specific primers corresponding to each virus. PCR products were cloned into pBluescript. Nucleotide sequences were determined by the dideoxy nucleotide chain termination method.

Results and conclusions
The nucleotide sequences of TYLCV-Ng and TYLCV-Sz are closely related to isolates reported from Israel. The nucleotide sequence homologies between TYLCV-NG and TYLCV-1s (Genbank accession no. X15656), TYLCV-SZ and TYLCV-1s mild isolate (X76319) are 98 and 97%, respectively. The amino acid sequence homologies of coat protein (CP) genes among these four isolates are 96-99%, indicating that these four isolates have a very close relationship. This conclusion was confirmed by phylogenetic analysis. TbLCV-Eu and TbLCV-Hs were considered to have a monopartite genome composed of six ORFs. Two of them [V1(CP), V2] are virion-sense and the other four ORFs (C1-C4) are complementary-sense. ORFs C1 of the two viruses a code a replication-associated protein with putative DNA-nicking domains (motif I-III ) as described [2]. TbLCV-Eu and TbLCV-Hs are most closely related to each other compared with other Old World subgroup 19 geminiviruses, although the amino acid sequence homologies of CP, ORFs V2 and C1-C4 between them are 83, 83, 85, 66, 75 and 79%, respectively. These rather low homologies suggest that some evolutionary diversity is occurring in TbLCVs. TbLCVs and TYLCVs are not closely related in the nucleotide sequences and are confirmed to be separate species.

References
1. Briddon RW, Markham PG, 1994. Molecular Biotechnology 1, 202-205.
2. Faria JC, Gilbertson RL, Hanson SF et al., 1994. Phytopathology 84, 321-329.