Assessment of the variability of different isolates of Watermelon silver mottle virus occurring in Taiwan

• Main Authors: Min-Hsun Chung, 鍾旻汛
• Other Authors: Shyi-Dong Yeh
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/30960010391984247395

碩士 === 國立中興大學 === 植物病理學系 === 90 === Abstract Watermelon silver mottle virus (WSMoV), a member of the genus Tospovirus, is the most important pathogen for watermelon and other cucurbits in Taiwan. The complete nucleotide sequences of genomic RNAs of WSMoV have been previously determined. Two pairs of degenerate primers targeting the conserved regions of L RNA were designed. The degenerate primer pairs not only useful for detection of tospoviruses in five different serogroups, but also can be used for efficient detection of field samples infected with WSMoV and TSWV. In this investigation, sixty-two samples were collected from watermelon at Chiding (Hsinchu City) and Dadu (Taichung County), melon at Luenbei (Yunlin County), calla lily at Beidou (Changhua County), watermelon in Tainan, and tomato in Tainan. Twenty-eight isolates were positive in ELISA using WSMoV polyclone antiserum. A 810-bp fragment was amplified from all the isolates by RT-PCR with the L RNA degenerate primers. A 689 and a 828-bp fragments were amplified by RT-PCR with two pairs of WSMoV N gene-specific primers from all the isolates except the calla lily isolate. Sequence analyses of the N genes revealed that the diversity of nucleotide sequences were from 98.3% to 99.5% and of protein sequences from 98.2% to 100%, indicating that the N gene sequences of these isolates share a high degree of similarity. In this investigation, the symptoms caused by calla lily isolate on Chenopodium quinoa plants were different from the control, while the others were quite similar. The calla lily isolate was detected by RT-PCR based on total RNA with degenerate primer pairs of L RNA but not with WSMoV N gene specific primer pairs. Furthermore, the calla lily isolate was serologically related to the N protein of WSMoV as reflected in positive reactions in ELISA and Western blot using WSMoV antiserum. To further identify the calla lily isolate, the dsRNA was isolated and its pattern was shown similar to that of WSMoV. A degenerate primer pair of L RNA and a degenerate primer pair designed targeting the 3' terminal conserved sequences of S RNA were used in RT-PCR. A 810- and a 750-bp DNA fragments were amplified and sequence analyses revealed that the diversity of nucleotide sequences with L gene and N gene of WSMoV were 81% and 40%, respectively. Thus, from the results of host reactions, serological relationships, RT-PCR, electron microscopy, and dsRNA analysis, it is suggested that the calla lily isolate is a new tospovirus. However, the taxonomy of the calla lily isolate will be determined by the further analysis of the N gene. The present study provided a preliminary account of genetic variability of WSMoV population occurring in Taiwan. Also, results of this study provide basic information for the control of WSMoV in Taiwan by the transgenic approach.