Characterization, complete genome sequence and genetic organization of Broad bean wilt virus-2 isolated from lisianthus in Taiwan

• Main Authors: Bing-Nan Shen, 沈炳男
• Other Authors: Fuh-Jyh Jan
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/80182787402811227994

碩士 === 國立中興大學 === 植物病理學系所 === 96 === Lisianthus [Eustoma grandiflorum (Ref.) Shinners] is a popular herbaceous ornamental plant worldwide. In 1996, a virus isolate LV-5 was obtained from lisianthus plants showing chlorotic and necrotic spots on leaves in Changhua, Taiwan. The virus was previously identified as a potential isolate of Broad bean wilt virus-2 (BBWV-2) since it positively reacted with a commercial polyclonal antibody against BBWV-2. The objectives of this study were to provide the molecular evidence to confirm that the virus isolate LV-5 is indeed a BBWV-2, to clone and sequence the genome of this virus, to characterize the biological properties and generate polyclonal antibody against this virus. Virus particles were purified from infected Nicotiana benthamiana plants and 28 nm isometric virions were observed by an electron microscopy. Polyclonal antibody was produced by immunizing the rabbit with purified virus, and the ability to detect BBWV-2 isolate LV-5 was demonstrated by western blotting and ELISA. The host range reactions test showed that 17 plant species belonging to 4 families were susceptible to BBWV-2 isolate LV-5. Coat protein (CP) genes of LV-5 were amplified by RT-PCR and then cloned and sequenced. Comparisons of the nucleotide sequence of large-CP (L-CP) and small-CP (S-CP) showed that LV-5 shared 77.6-92.3% and 77.1-93.9% identities, respectively, to those of other eighteen BBWV-2 isolates. Comparisons of the amino acid sequence of L-CP and S-CP showed that LV-5 shared 84.8-97.5% and 88.8-98.0% identities, respectively, to those of other BBWV-2 isolates. The L-CP of LV-5 has 61.5-62.3% nucleotide and 62.7-64.4% amino acid identies with those of BBWV-1 isolates. The S-CP of LV-5 has 60.5-62.4% nucleotide and 58.4-59.9% amino acid identities with those of BBWV-1 isolates. Near full-length genomic sequences was obtained via genome walking techniques and the 5´ terminal sequences were obtained from RT-PCR with oligo-d(T) as primers using polyadenylated dsRNA as template. Excluding the 3´ terminal poly-A tails, the RNA 1 and 2 are 5947 nt and 3555 nt, respectively. All genes on both RNAs were determined according to the proposed protease cleavage sites. Comparison of the full-length sequences showed that the LV-5 isolate shared 78.6-93.0% identity on RNA 1 and 78.7-92.0% on RNA 2 to those of other seven published BBWV-2 isolates. Phylogenetic analyses based on the full-length nucleotide sequences of RNA 1 or RNA 2 and the amino acid sequences of two coat proteins showed that LV-5 isolated from lisianthus in Taiwan was closely related to the BBWV-2 IP isolate infecting pepper in Japan. Taken together, our results provide the molecular evidence to confirm that virus isolate LV-5 from lisianthus in Taiwan is an isolate of BBWV-2.