應用Real-Time RT-PCR技術研究木瓜輪點病毒在木瓜 寄主體內的分佈、移動以及增殖動態

Abstract

木瓜輪點病由木瓜輪點病毒 (Papaya ringspot virus, PRSV) 所引起，是木瓜重要的病害之一。台灣目前的PRSV依據在木瓜上所引起的病徵不同，再細分為各種不同系統，包括可引起葉片產生嚴重嵌紋的嚴重嵌紋系統 (severe mottling, SM strain )、造成葉片嚴重嵌紋且畸形扭曲的畸型系統 (deformation, DF strain)，以及造成葉片嚴重嵌紋且伴隨有壞疽病斑的嚴重嵌紋壞疽系統 (severe mottling with necrosis, SMN strain); DF為目前台灣田間最常見之系統，而SMN會在冷熱交替之際造成快速萎凋的病徵，被視為最具摧毀潛力的系統。本論文嘗試在不同品種的木瓜上接種不同系統的PRSV (包括DF與SMN系統)，以即時定量反轉錄聚合酶連鎖反應 (Real-Time RT-PCR) 的分子檢測技術追蹤病毒在木瓜在根、莖、葉等部位上增殖情形，並且進一步做定量比較。除了可得知PRSV在寄主體內的真正分佈狀況外，也能推測病毒移動的趨向，且掌握到各病毒系統在木瓜寄主最終之飽和病毒量，更深一層了解各不同病毒系統的病原性。此外，本論文也以不同木瓜品系進行試驗，比較PRSV在台農二號、紅妃、台大八號等不同品系木瓜的增殖動態與分佈情況。本論文以DF與SMN二系統進行接種實驗，並定期追蹤病毒的增殖動態。將台農二號、紅妃、台大八號三個木瓜品種個別接種DF及SMN系統後，其移動的路徑皆頗為一致，發現PRSV自接種葉感染後會先向根部移動，逐步拓展至莖部，爾後再向上轉移到葉部。以PRSV/DF為接種源時，台大八號品種在寄主植株各部位皆最先被測出病毒訊號，如根部與莖部平均接種後第二天即可測出，葉部則是在接種後4天可以測到，顯示病毒在台大八號上的移動十分迅速；但到達葉部後病毒增殖並不順利而一直維持於較低濃度 (約103 copies)；台農二號各部位都較晚被偵測到，根部與莖部分別在接種後4天與8天被測到，10天後病毒才達葉部，但後勢看漲，24天後在葉部之病毒量遠高於其他品種 (約107~8 copies)；紅妃的根部、莖部與葉部分別在接種後7、8與8天被測到，24天後在葉部之病毒量約為104 copies。以PRSV/SMN為接種源時，台大八號根部與莖部也是在接種後第二天即可測出，葉部則是在接種後7天可以測到，24天後在葉部測到的病毒量仍不高 (約103~4 copies)；台農二號根部與莖部分別在接種後8與4天被測到，10天後病毒才達葉部， 24天後在葉部之病毒量明顯提升，30天後可達106 copies；紅妃的根部、莖部與葉部分別在接種後8天、8天與4天被測到，24天後在葉部之病毒量約為104 copies。就病毒在植株體內各部位的PRSV定量偵測結果來看，根部的PRSV累積量通常要高於或相似於葉部，莖部病毒量略低於根部但差距並不明顯，由此可見根部是PRSV相當重要的繁殖部位。新育成之耐病品種台大八號接種PRSV/DF時，根部病毒量最高可達106 copies，但轉移至葉部增殖時只能達到約103 copies，與感病的台農二號相比低了約104~5倍，顯示病毒在台大八號的葉部有明顯被壓制，推論可能是其耐病原因之ㄧ；台大八號接種PRSV/SMN時，根部病毒量最高可達107~8 copies，但轉移至葉部增殖時只能達到約103~4 copies，情況與DF類似，但可看出台大八號對SMN的壓制力稍差。此外，比較傳統反轉錄聚合酶連鎖反應 (RT-PCR) 技術與Real-time RT-PCR技術偵測結果，可以發現Real-Time RT-PCR不但可以較早或同時偵測出病毒存在，且提供了相當精準的相對量值，了解寄主體內病毒之增殖動態，為未來研究木瓜輪點病毒及相關研究分析不可或缺的工具之一。

Papaya ringspot , caused by the Papaya ringspot virus (PRSV), is one of the most important diseases in papaya. PRSV is divided into three major strains distinguished by the symptoms on leaves: severe mottling (SM), deformation (DF), and severe mottling with necrosis (SMN). In this study, three papaya cultivars (lines) were inoculated with PRSV (DF and SMN strain), then the Real-Time RT-PCR assay was used to perform quantitative detection to track the distribution, migration, and propagation of each PRSV in papaya hosts. Three papaya cultivars (lines) were used including Tainung No.2 (TN2), Red Lady (RL), and National Taiwan University Hybrid No.8. (NTU8). After inoculation with the DF strain of PRSV (PRSV/DF), viruses could be detected in roots, stems and leaves of NTU8 within 2 days, which is earliest among the three cultivars. Viruses were found in NTU8 leaves within 4 days post-inoculation, indicating that the viruses moved very fast in NTU8, although they did not replicate well and remained at low quantity (103 copies). PRSV/DF was monitored in roots, stems and leaves of TN2 4, 8 and 10 days after inoculation respectively; the virus quantities in leaves of TN2 reached to a peak (107~8 copies) 24 days later, which is the highest compared to RL and NTU8 . For the RL cultivar, viruses were detected in roots, stems and leaves at 7, 8, and 8 days post-inoculation respectively, and the peak quantity in leaves 24 days after inoculation reached 104 copies. In the inoculation tests with the SMN strain of PRSV (PRSV/SMN), NTU8 exhibited detectable virus levels in roots, stems and leaves 2, 2 and 7 days after inoculation respectively, and the virus quantity remained (103~4 copies) at 24 days later. PRSV/SMN in TN2 was detected in roots and stem after 8 and 4 days respectively, and reached into leaves 10 days after inoculation, where quantity could reach as high as 106 copies after 30 days. The roots, stems and leaves of RL showed detectable virus at 8, 8 and 4 days after inoculation, with quantities at about 104 copies in the leaves 24 days later. In roots usually accumulate more or the same amounts than in leaves, and stems gave rise to slightly lower quantity of viruses than in roots as a results of comparative quantitative detection among roots, stems and leaves no matter what cultivar of papaya was used. It seems that roots are very important for PRSV to propagate. The new bred papaya cultivar tolerant to PRSV, NTU8, constantly showed a good replication of PRSV/DF at 106 copies in roots, but a lower amount of 103 copies in the leaves. TN2, the susceptible cultivar to PRSV, showed more viruses in the leaves approximately 104~5 times than NTU8. This is probably one of the factors resulting in the tolerance of NTU8. When NTU8 was inoculated with PRSV/SMN, it had the virus quantity of 107~8 copies in roots, but only 103~4 copies in leaves; which is similar to the case of PRSV/DF. However, NTU8 seems to have slightly lower tolerance to PRSV/SMN. In this thesis, in addition, the Real-Time RT-PCR method performed its better sensitivity and could detect these viruses at the same time or earlier than the conventional RT-PCR method. Furthermore, this method also provides a precise relative quantification for PRSV and it is helpful to understand how the virus propagates in the host, and will be valuable for future ecological study and control of PRSV.