Characterization of Broad bean wilt virus-2 isolated from Salvia dorisiana STANDL

• Main Authors: Chen-An Lin, 林建安
• Other Authors: Hui-Liang Wang
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/62192110837221213371

碩士 === 國立高雄師範大學 === 生物科技系 === 95 === Fruit sage (Salvia dorisiana) seedlings with mosaic symptom were found from the herb nursery in Nantou. The virus isolate was obtained from three successive single lesion isolations on Chenopodium quinoa and subsequently tested for its infectivity on 37 plant species from 17 families by mechanical inoculation. Three species in Chenopodiaceae including Chenopodium amaranticolor, C. quinoa and Spinacia oleracea and one of the plants from Amaranthaceae, Gomphrena globosa, could be infected. Examination of preparations of purified virus by electron microscopy revealed some spherical particles of 26 nm in diameter were observed. The thermal inactivation point of the virus was 55 ℃ to 65 ℃, dilution end-point was 10-3 to 10-4 and the longevity in vitro of the infectious sap was 8 days at room temperature. Differential centrifugation followed by cesium sulphate (Cs2SO4) density gradient centrifugation of preparations from tissues of infected C. quinoa were conducted. Two white bands of virus were observed below top surface 4.8 cm and 5.7 cm of the centrifuge tube after purification. The virus had a maximal absorption at 258 nm and a minimal absorption at 237 nm. Amax/Amin and A260/A280 ratio were 1.62 and 1.73, respectively. The yield of purification from 100 g tissue of C. quinoa were 2.08 mg. When thirteen kinds of antisera were used in direct or indirect ELISA, it only reacted to the antiserum to Broad bean wilt virus 2 (BBWV-2). Analysis by SDS-polyarylamide gel electrophoresis of purified virus preparations showed that the viron contained two structural polypeptides with 21 and 43 kDa relative molecular weights, respectively. Cloning and sequencing part of viral RNA 2 by RT-PCR (reverse transcription- polymerase chain reaction) with degenerate and specific primer pairs obtained 1833 nucleotides encoding a large coat protein (LCP) and a small coat protein (SCP). The LCP gene with 1206 nucleotides encoded a 44.3 kDa protein of 402 amino acids. The SCP gene with 594 nucleotides encoded a 22.2 kDa protein of 197 amino acids. The nucleotide and amino acid sequences of LCP and SCP genes were compared with BBWV sequences in NCBI database by DANSIS and VECTOR NTI software. The highest nucleotide identity of LCP and SCP genes were both 95% with Patchouli mild mosaic virus (accession number AB11007). The lowest identity of LCP and SCP genes were 61% and 60% with PV176 strain of BBWV-1 (accession number AB018703), respectively. The highest amino acid similarity of LCP and SCP genes were 98% and 97% with MB7 isolate of BBWV-2 (accession number AB013616), respectively. The lowest similarities of LCP and SCP genes were 64% and 59% with PV131 strain of BBWV-1 (accession number AB018702), respectively. Phylogenetic tree obtained from the alignments of the LCP and SCP genes amino acid sequences showed that the virus was a strain of BBWV-2 and named BBWV2-FS. This is the first report of natural occurrence of BBWV to cause a disease on fruit sage in Taiwan.