Strain-differentiation of Citrus tatter leaf virus (CTLV) and development of a rapid method for CTLV detection

• Main Authors: Yu-Hsuan Lin, 林育萱
• Other Authors: Ting-Husan Hung
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/87337531614154677992

碩士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 92 === Citrus tatter leaf disease is one of the important systemic diseases of citrus. It is caused by Citrus tatter leaf virus (CTLV), which is categorized into the genus Capillovirus consisting of a single-stranded, positive-sense RNA genome packaged in circa 650 nm flexuous particles. CTLV is transmitted by vegetative propagation and / or mechanical inoculation. The virus is usually latent in most commercialized citrus cultivars, but it induces dwarfing, distortion, mottling and “tatter leaf” in the infected leaves of susceptible citrus hosts such as Kalpi lime (Citrus excelsa) and Rusk citrange (Poncirus trifoliate × C.sinensis). It may also incite the “bud-scion crease” symptom, which retards citrus growth and sometimes causes breakage at the bud union when the strong wind blows. Su et al. investigated the infection rate for CTLV in citrus plants cultivated in Taiwan. They discovered that approximately 70%~80% of the collected samples were positive for CTLV in the bioassay with “Rusk” indicator plants and CTLV has distributed throughout Taiwan. Recently, the “bud-scion crease” symptom was found in several citrus cultivars with citrange or citrumelo rootstocks grown in the citrus nursery foundation of Chiayi Agricultural Experiment Station (CAES), Taiwan Agricultural Research Institute. The results of bioassays demonstrated that they had been infected by CTLV. This study is dedicated to categorize the different CTLV strains on the biological and molecular level for understanding of the pathological characters of CTLV. In the biologically experimental results, four distinct isolates including LCd-NA-1, Kq-6-2-46, Sat-HY-2 and Cal-KS-1 isolates can be differentiated based on their induced symptoms in the leaves of Rusk citrange. In addition, four CTLV isolates also caused different foliar symptoms in Chenopodium quinoa, the local lesion host grown in a greenhouse under temperature control at 30℃and 22℃. To study the molecular characteristics and genomic structures of two Taiwanese isolates, LCd-NA-1 (from Luchen sweet orange) and Kq-6-2-46 (from kumquat swingle), their full genomic sequences (totally 6496 bases) have been determined and posted in GenBank (accession number AY646511). Alignment of full nucleotide sequences among LCd-NA-1, Kq-6-2-46 and several relative Capilloviruses indicated that the sequence homology is 95% between LCd-NA-1 and Kq-6-2-46; it is about 81% between the Taiwanese CTLV and the Japanese CTLV；and it is about 80% between the Taiwanese CTLV and the other Capilloviruses. On the other hand, a reliable and rapid assay based on the reverse transcription-polymerase chain reaction (RT-PCR) technique was developed for CTLV detection. Four out of ten RNA extraction methods were selected for efficient RT-PCR assays, and the “TRIzol Reagent method” was proven to be the most economic, sensitive and reliable among four feasible methods. Two primer pairs, named CTLV 636 and CTLV 527, were selected to establish a rapid and accurate detecting technique for CTLV with optimized one-step RT-PCR assays. Primer pair CTLV 636, designed from the common region of CTLV coat protein gene, is suitable for the general CTLV RT-PCR detection; Primer pair CTLV 527, designed from the varible region of CTLV coat protein gene, is suitable for differentiating Taiwan CTLV strains from foreign strains. A 636-bp fragment could be amplified from the diseased samples above by RT-PCR with primer pair CTLV 636. Our RT-PCR assay was applied to detect CTLV in many citrus cultivars in the CAES foundation. CTLV was detected in Luchen sweet orange, Valencia sweet orange, Kumquat swingle, Murcott, Minneola tangelo and Ortanique tangor. These amplified fragments were further cloned and sequenced. A phylogenetic tree was obtained according to the alignment of 636 nucleotide sequences among several domestic and foreign CTLV isolates. The viruses in the same geographical regions have high degree of sequence identity. This study provides biological and molecular evidences to demonstrate that CTLV has diverse strains in nature, and is an important step toward understanding of citrus tatter leaf disease and the developed RT-PCR assay is absolutely helpful in the progress of pathogen-free nursery policy of citrus in Taiwan.