仙人掌X病毒與紅龍果X病毒於紅龍果植株之感染和體內分布

Abstract

紅龍果 (Pitaya)多指屬於仙人掌科 (Cactaceae)、三角柱屬 (Hylocereus)的紅皮白肉紅龍果 (H. undatus)，以及有許多雜交品系的紅皮紅肉品種 (Hylocereus spp.)。台灣已知感染紅龍果的病毒皆為Potexvirus屬：仙人掌X病毒 (Cactus virus X, CVX)、紅龍果X病毒 (Pitaya virus X, PiVX)與蟹爪蘭X病毒 (Zygocactus virus X, ZyVX)，且田間調查發現，幾乎全台皆有這些病毒分佈。本研究主要目的為建立CVX與PiVX病毒接種系統以及研究病毒在植株體內移動與分佈情形。由於目前沒有將紅龍果病毒接種至紅龍果植株的有效方法，因此希望能夠建立一套高成功率之接種系統，以利後續實驗進行。前人文獻指出仙人掌科植株對於受傷反應會快速形成隔離組織，可能使病毒接種不易成功；因此我們選擇白肉紅龍果實生苗為材料，應用金剛砂接種子葉，分析病毒的感染情形。當以ELISA進行檢測時，子葉單獨接種CVX 9日後可獲得80%受CVX感染的葉狀枝；而單獨接種PiVX時，於11日後，有40%葉狀枝受PiVX感染。藉由子葉接種系統觀察CVX在白肉紅龍果實生苗的植物體內感染與移動的變化情形，發現在接種後3日到12日為病毒進行長距離移動的關鍵期。在CVX與PiVX病毒濃度相同時，不論是單獨或複合接種，從MNC RT-PCR結果發現CVX在紅龍果實生苗中的移動能力比PiVX好。在複合接種下，PiVX的感染率較單獨接種佳，暗示PiVX在移動上有可能受到CVX的幫助。在病毒總濃度相同下，以MNC RT-PCR檢測單獨或複合接種之結果顯示，3日後於接種的子葉可100%測得病毒感染；而從第9天的結果發現，PiVX的感染情形在單獨接種之下較佳，推測PiVX對PiVX在移動上的協助大於CVX對PiVX的協助。依據組織轉印免疫分析法之結果，感染CVX或是PiVX的植株，無論在莖頂端或根部皆可測得病毒訊號。以簡單組織固定法配合免疫組織化學染色法，目前可在罹病株的不含莖頂分生組織的周圍細胞測得較明顯的紅龍果病毒訊號，顯示CVX與PiVX可系統性感染紅龍果，但可能無法入侵分生組織。

Pitayas usually indicate the fruit crops belonging to the genus Hylocereus (Cactaceae) and their fruits can be grouped into red skin white flesh (H. undatus) and red skin red flesh (Hylocereus spp.). Cactus virus X (CVX), Pitaya virus X (PiVX) and Zygocactus virus X (ZyVX) are known pitaya viruses in Taiwan and they all belong to the genus Potexvirus. Field surveys showed that these viruses exist in most areas in Taiwan. The purposes of this study are to build up pitaya virus inoculation system and to investigate the movement and distribution of the viruses. Because there is no efficient inoculation method for pitaya viruses, we want to establish an inoculation system with high successful rate for further studies. Based on previous studies, cactus plants quickly form barrier tissues after they are wounded. This property may interfere with the success of virus inoculation. Therefore, pitaya (H. undatus) seedlings were chosen as plant materials, the cotyledons were mechanically inoculated with the help of carborundum, and then viral infection was investigated. By ELISA test, 80% of CVX-infected phyllocaldes were obtained after 9 days of CVX single inoculation but only 40% of PiVX-infected phyllocaldes were acquired after 11 days of PiVX single inoculation. Using cotyledon inoculation method to study CVX infection and movement inside the pitaya seedlings, the result indicated that the period between 3 to 12 days post inoculation is the key stage of CVX long-distance movement. When inoculated with the same concentration of CVX and PiVX, CVX showed better movement ability than PiVX in single- and mixed-inoculation according to MNC RT-PCR. PiVX infection rate was higher in mixed-inoculation than in single-inoculation. The result suggested that CVX may help PiVX move inside the plant. When single- or mix-inoculated with the same total virus concentration, the results of MNC RT-PCR revealed cotyledons were 100% infected after 3 days; PiVX showed higher infection rate in single inoculation after 9 days. Therefore, it is proposed the assistance in PiVX movement by PiVX might be better than by CVX. Based on tissue blot immunoassay, viral signals could be detected in the shoot tips and roots of CVX- or PiVX-infected pitaya seedlings. By using simple fixation combined with immunohistochemistry, we observed obvious virus signals around the shoot meristem of virus-infected pitaya seedling but not in the central of shoot meristem. These results demonstrated that CVX and PiVX can systemically infect the pitaya seedling but maybe they cannot invade the shoot meristem.