T. Ito1, T. Ogawa1, K. Samretwanich2, P. Sharma1 and M. Ikegami1*
1 Department of Life Science, Graduate School of Agricultural Science, Tohoku University, 1-1, Tsutsumidori-Aamamiyamachi, Aoba-ku, Sendai, Miyagi 981-8555, Japan
2 National Research Council of Thailand, 196 Phaholyothin Road, Chatuchak, Bangkok 10900, Thailand
Accepted: 09 Oct 2007
Yellow leaf curl disease causes heavy losses to cucurbit crops in Thailand. Previously, we have detected Tomato leaf curl New Delhi virus from infected cucumber (Cucumis sativus), wax gourd (Benincasa hispida), cantaloupe (Cucumis melo) and muskmelon (Cucumis melo) plants in Thailand (Samretwanich et al., 2000a, b). Pumpkin (Cucurbita pepo) is also severely affected by this disease. Three pumpkin plants exhibiting leaf curling and yellowing symptoms (Fig.1) were collected from Kamphaensaen, Nakom Pathom in Thailand during 2001.
Total DNA was extracted from leaves and PCR was conducted using begomovirus specific degenerate primers UPV1 and UPC2 designed to amplify DNA-A (Briddon and Markham, 1994). A PCR product of approximately 2.7kbp was obtained, cloned and sequenced. To obtain the full DNA A sequence a pair of degenerate primers with an XbaI site (underlined) were designed to the partial sequence (virion-sense primer 5′-TCTAGAACGTCTCCGTCTTTG-3′; complementary-sense primer 5′-TCTAGAAATGGGGTGTTTTCC-3′) and used in PCR to amplify a product of approximately 2.8kbp, which was cloned and sequenced (2736nt, Accession No. AB330078). Efforts to detect DNA-B component associated with the virus by PCR using primers PCRc1 and PBL1v2040 (Rojas et al., 1993) were unsuccessful. The DNA-A contained the five open reading frames typical of begomoviruses, two in the virion sense and three in the complementary sense. Comparison of the complete nucleotide sequence of this begomovirus with those of full-length begomoviral DNA-As available in GenBank showed the highest nucleotide sequence identity (95 and 89%) with Squash leaf curl China virus (SLCCNV, Accession No. AB027465) and Squash leaf curl Philippines virus (SLCPHV; Accession No. AB085793), respectively. For ORF V1 (coat protein), this begomovirus had the highest amino acid sequence identity with its counterparts in SLCCNV (97%) and SLCPHV (97%). Therefore the present virus is an isolate of SLCCNV, which we designate SLCCNV-Thailand. This is a first report of SLCCNV infecting pumpkin in Thailand.
This work was partly supported by the Grant-in-Aid for Academic Frontier Promotion programme and Grant-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Briddon RW, Markham PG, 1994. Universal primers for the PCR amplification of dicot-infecting geminiviruses. Molecular Biotechnology 1, 202-205.
Rojas MR, Gilbertson RL, Russell DR, Maxwell DP, 1993. Use of degenerate primers in the polymerase chain reaction to detect whitefly-transmitted geminiviruses. Plant Disease 77, 340-347.
Samretwanich K, Chiemsombat P, Kittipakorn K, Ikegami M, 2000a. Tomato leaf curl geminivirus associated with cucumber yellow leaf disease in Thailand. Journal of Phytopathology 148, 615-617.
Samretwanich K, Chiemsombat P, Kittipakorn K, Ikegami M, 2000b.Yellow leaf disease of Muskmelon from Thailand caused by Tomato leaf curl virus. Plant Disease 84, 707.
©2007 The Authors