木瓜輪點病毒兩系統在不同木瓜品種中之病理性差異與交互作用

Abstract

木瓜輪點病毒（Papaya ringspot virus = PRSV）是木瓜栽培中主要的限制因子，番木瓜罹病植株的葉片會產生嵌紋、黃化等病徵，果實出現輪點病斑，影響果實外觀、產量與品質。依據葉片上所引起病徵做區分，PRSV可分為畸形系統（deformation , DF strain）、嚴重嵌紋壞疽系統（severe mottling with necrosis, SMN strain）與嚴重嵌紋系統（severe mottling, SM strain）。畸形系統為目前田間最常見的系統，但嚴重嵌紋壞疽系統則最具摧毀性。本實驗擬透過病徵表現與植株內病毒增殖情形作為依據，綜合病理性與分子性的試驗證據，建立一套完整的木瓜寄主對PRSV抗（耐）感病性的評估方法，實驗過程之中配合應用即時定量反轉錄聚合酶連鎖反應（real-time RT-PCR）的追蹤，比較受試品系與對照的常用品系（如台農一號、台農二號、紅妃等）之間病毒增殖動態之差異，以協助新育成木瓜品系之篩選。此外，以同時接種 DF與SMN系統的病毒感染方式、或兩者一前一後的感染方式進行試驗，藉由具有系統特異性的real-time RT-PCR定量偵測法來追蹤不同病毒系統的增殖狀態，探討兩系統在木瓜寄主中的交互作用，裨益瞭解受試品系對兩個病毒系統複合感染的抗（耐）病性。由本論文的挑戰接種試驗結果得知受試之三新品系（10A、10B與10F）皆對DF系統有耐病性，但只有10A品系對SMN系統有耐病性，另兩品系對SMN則較感病。RT-PCR均偵測到受試木瓜三品系均可被PRSV感染，而real-time RT-PCR偵測結果更發現新品系木瓜體內病毒量高於一般常見的木瓜品種約10倍以上，顯示病毒在新品系中增殖狀況更佳，但是外表病徵輕微而病徵指數低，表示其耐病性相當良好，PRSV的大量複製並未導致嚴重的病徵產生。另一方面，試驗發現SMN系統對商業品種台農二號與紅妃適應力好，體內病毒量高，田間栽培二品種有利於SMN系統生存。病毒系統之交互作用的試驗部份，同步與非同步接種兩系統病毒後，發現先接種的病毒主導病徵發展，以台大八號(NTU 8)進行先接種DF、後接種SMN感染試驗中，先接種SMN系統會被DF系統追上或拉近差距；反之，先接種DF、後接種SMN時，後接種的SMN系統無法追上原先DF系統病毒量；同步接種二病毒系統時寄主體內兩病毒系統互相競爭，後期DF系統取得優勢。由此看出DF系統在田間似乎仍較SMN系統優勢，但仍需注意未來PRSV系統之間的演化發展。本研究應用real-time RT-PCR進行PRSV的定量偵測，綜合病毒增殖與病徵發展的兩項資訊，可提供評估新品系木瓜抗（耐）病性時之重要參考資訊；複合感染試驗的結果也顯示，台灣木瓜新品種台大八號目前耐病性仍很穩定，唯因應PRSV系統的長期演化，長期的耐抗病育種工作仍應持續。

Papaya ringspot virus (PRSV) is one of the limiting factors in the papaya industry. The infected papayas produce several symptom such as mosaic, yellowing, ringspot in fruit, which causes tremendous economic losses and poor quality of fruits. According to the symptoms developed on leaves, PRSV was categorized into three strains: SM(severe mottling), DF(deformation),and SMN(severe mottling with necrosis)strains. The DF strain is predominated in the field at present time, and the SMN strain induce most destructive symptom in papayas. This study we dedicated to investigate the correlation between the severity of symptom and replication of virus, and develop a evaluation method to evaluate the resistance or tolerance of new papaya cultivars. The RT-PCR and real-time RT-PCR assays were used in this study to monitor the fluctuation dynamic of virus replication for comparative study of tolerance between new developed papaya lines and commercial cultivars. In addition, the simultaneously or asynchronously inoculation experiment test with the DF and SMN strains to were also conducted to investigate the interaction between these 2 strains in either new line or commercial cultivars of papaya based on the real-time RT-PCR assays with train-specific primers. The result might be able to know the co-infection effect of the DF and SMN strains in various papaya cultivars(lines). The inoculation tests showed that all of 3 newly bred papaya lines(10A, 10B, and 10F) were tolerant to the DF strain without appearing apparent symptoms. However, only the 10A line is tolerant to the SMN strain. The real-time RT-PCR assays demonstrated the amounts of replicated virus in newly bred lines was 10 times higher than those in commercial cultivars. It means revealed that these newly bred papaya lines are actually better host for PRSV-replication though they do not produce symptoms showing their tolerance. The data also indicated that the SMN strain replicated very well in either Tainung 2 (TN 2) and Red-Lady (RL) cultivars, which means TN 2 and RL probably provide better conditions for survival of PRSV-SMN. In the asynchronously inoculation tests with both DF and SMN strains, all of infected papayas by PRSV-DF before PRSV-SMN (2-week interval) developed the symptoms similar to those induced by PRSV-DF whereas all of infected papayas by PRSV-SMN before PRSV-DF developed the symptoms similar to those induced by PRSV-SMN. It presented that the earlier invading virus strain determines the symptom-type. The DF-induced symptoms could be visualized when the papaya hosts were co-infected by the DF and SMN simultaneously. In the inoculation tests of the NTU8 papaya, the real-time RT-PCR assays demonstrated that the later invading PRSV-DF could replicate well and reach or exceed the amount of earlier infecting PRSV-SMN whereas the later invading PRSV-SMN could not reach the amount of earlier infecting PRSV-DF. These two strains competed each other when they simultaneously co-infected a papaya host, and the DF strain gradually became prevailing. This study reveals that the DF trains seemed to be more predominant than the SMN strain in the field, and we should pay more attention to the evolution of various PRSV strains. The investigation of symptomatology and quantitative detections of PRSV based on real-time RT-PCR assays provided important data to evaluate the resistance or tolerance against PRSV for newly bred papaya lines. The newly developed papaya cultivar tolerant to PRSV, NTU 8, showed its stable tolerance in the co-infection trials with both DF and SMN strains. However, the continuous breeding of papaya should be conducted to cope with the evolution of various strains of PRSV.