木瓜畸葉嵌紋病毒感染性選殖株之構築及與木瓜輪點病毒之交互作用

Abstract

木瓜為台灣重要的熱帶經濟果樹之一，台灣木瓜產業最大的限制因子為木瓜輪點病，但近年來發現另一木瓜病毒－木瓜畸葉嵌紋病毒 (Papaya leaf distortion virus， PLDMV)，其病徵、生態地位、傳播媒介與木瓜輪點極為相似，因此被認為極有潛力成為台灣木瓜產業新的限制因子，迫切需要進一步研究。本研究自屏東長治分離到新的PLDMV分離株（PLDMV-CZ），經接種至木瓜後，其病徵初期為葉片斑駁、葉脈黃化、透化，嚴重時造成嚴重嵌紋並使葉片嚴重變形。經寄主範圍之試驗，發現除木瓜外無法感染其他十七種測試植物，特性與先前台灣其他發現的分離株相似。經全長病毒基因體解序，PLDMV-CZ全長共有10154個核苷酸，與先前發表之台灣高樹分離株的全長相似度為95.5%，與台灣大里分離株、日本J56P分離株相似度則為94.5%。由於PLDMV未能發現單斑寄主，對於其病理特性難以完整釐清，因此本研究擬藉由建構PLDMV感染性選殖株來得到純系病毒，並可應用於研究病毒分子特性。本論文分別採取「一次法」及「分段法」來建立PLDMV-CZ全長cDNA選殖株；「一次法」乃利用管柱式的純化套組抽取植物全RNA後，設計引子對以進行反轉錄聚合酶連鎖反應(RT-PCR)，直接增幅出帶有T3 promoter及poly A尾端的PLDMV全長cDNA（10.2kb），以載體pCC1進行選殖，得到三個選殖株；「分段法」將PLDMV全長分成三個互相重疊的片段，以RT-PCR分別增幅出3條cDNA，經選殖、剪接與黏合後得到全長cDNA選殖株，共計3株。所有選殖株經胞外轉錄後，將合成的RNA以機械方式感染木瓜幼苗，結果發現「一次法」所獲得的3個選植株中有1個可成功感染，「分段法」則三個選植株皆可感染，所有感染性選植株產生之病徵與野生病毒株皆無明顯差異。另外，為評估PLDMV的可能影響，本研究以同步、非同步接種PLDMV-CZ及木瓜輪點病毒畸型系統(PRSV-DF)兩病毒於台大八號雜交二代(NTU 8-F2)與台農二號(TN 2)兩種木瓜上，並以Real-time RT-PCR偵測兩病毒增殖動態，發現複合感染PLDMV-CZ與PRSV-DF之木瓜植株其病徵嚴重度似與單獨接種PLDMV-CZ時並無差異，而單獨感染PRSV-DF在NTU 8-F2木瓜到最後病徵有回復情形，但單獨感染PLDMV-CZ時則無。而病毒增殖動態中，兩病毒皆在接種後10天達即達到穩定，兩者病毒量約在10^7~10^8 copy numbers之間；同步接種時兩病毒量相當，不會互相影響；非同步接種時，後接之PLDMV-CZ在接種後16天達到穩定，PRSV-DF 26天才達到穩定，後接種之病毒不會影響先接種之病毒的增殖情形。此結果顯示PLDMV-CZ及PRSV-DF可能可以相互穩定共存於田間，且兩者無協力作用。另外本實驗中也觀察到PLDMV-CZ在木瓜寄主內之增殖情形比先前發現的高樹分離株(PLDMV-KS)為佳。以本實驗結果，PLDMV-CZ具有危害潛力，未來推出新品種時也應將PLDMV-CZ的危害性列入評估。

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.