Papaya leaf distortion mosaic virus infectious clone construction and interaction with Papaya ringspot virus

• Main Authors: Ping-Hu Wu, 吳秉祜
• Other Authors: Ting-Hsuan Hung
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/44435358560836725586

碩士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 100 === Papaya is one of important tropical fruits in Taiwan. Papaya ring spot caused by Papaya ringspot virus (PRSV) is a main limiting factor for the papaya industry. Papaya leaf distortion virus (PLDMV) is a newly emerged virus of papaya in Taiwan. The pathological characteristics of PLDMV such as symptoms, transmission and ecological niche were similar to those of PRSV. PLDMV is therefore considered to be a potential threat just as PRSV for papayas. A PLDMV isolate named PLDMV-CZ was collected from the papaya orchards in Chang-Chi, Pingtong, and it incited severe mottling, vein yellowing, vein clearing and leaf-distortion in papaya hosts in the inoculation tests. PLDMV-CZ could not infect 17 plants except the papaya, which presented the results similar to those PLDMV isolates shown in the previous studies. The sequencing data revealed that the whole genome of PLDMV-CZ consists of 10,154 nucleotides, and they are 95.5% identical to those of PLDMV-KS and 94.5% to those of PLDMV-DL and PLDMV-J56P (from Japan). There is no local lesion host for isolation of PLDMV, so the pathological characterists of PLDMV could not be easily clarified. Thus, this thesis was dedicated to construct infectious clones of PLDMV to obtain pure PLDMV isolates. Two methods were used for construction of infectious clones: one-step (direct) and traditional (splicing) methods. In one-step method, the full-length cDNA fragments of PLDMV-CZ with T3 promoter and polyA tail (totally 10.2 kb) were directly amplified by the reverse transcription-polymerase chain reaction (RT-PCR) with devised primer pairs from the total RNA extracts, and the amplified fragments were then cloned into vector pCC1. Three clones were obtained by the one-step method, and only one clone could successfully infect papaya after synthesizing artificial viral RNAs through in vitro transcript. In the traditional method, the full-length cDNA fragments of PLDMV-CZ were spliced from 3 overlapping amplified fragments of RT-PCR and they were then ligated to the cloning vectors. Three clones were obtained by the traditional method, and all of them were proven to be able to infect papayas. The symptoms induced by these infectious clones were indistinguishable from those caused by wild-type of PLDMV-CZ. In order to evaluate the infectious potential of PLDMV, simultaneously and asynchronous inoculation tests in NTU8-F2 and TN-2 papaya cultivars with PLDMV-CZ and PRSV-deformation strain (PRSV-DF) were conducted in this thesis. The results demonstrated that the symptoms simultaneously caused by PLDMV-CZ and PRSV-DF could not be distinguished from those caused by single inoculation of PLDMV-CZ. The NTU8-F2 papayas inoculated with PRSV-DF produced milder symptoms and finally showed healthy-looking, but the plants infected by PLDMV-CZ kept the apparent symptoms. The multiplicative dynamics of PLDMV-CZ and PRSV-DF were monitored by the realtime RT-PCR assays in the inoculation tests. The results showed that either PLDMV-CZ or PRSV-DF reached the maximan amount with 10^7-10^8 copy numbers 10 days post inoculation (dpi). On the other hand, the asynchronous tests presented that PLDMV-CZ reached the maximan amount 16 dpi when the plants were infected PRSV-DF 2 weeks before. PRSV-DF reached the maximan amount 26 dpi when the plants were infected PLDMV-CZ 2 weeks before. The asynchronous inoculation tests showed that the later invasive virus did not significantly affect the replication of previous invasive virus. It seemed that PLDMV-CZ and PRSV-DF could exist independently in the papaya hosts without interference each other, and they did not show synergistic symptoms when they co-infected papayas. PLDMVshould be considered as a potential threat to papaya industry especially when new cultivars were released.