Transmission ecology of Konjac mosaic virus in calla lily by aphids and virus rapid detection

彩色海芋 (Zantedeschia spp.) 屬於天南星科 (Araceae) 的觀賞植物，原產於非洲南部，90年代始從紐西蘭和荷蘭引進台灣，生產切花及盆花。彩色海芋因其繽紛多彩的佛焰苞，廣受到消費者的歡迎。然而，細菌性軟腐病與病毒病害一直是彩色海芋栽培上的主要限制因子。蒟蒻嵌紋病毒 (Konjak mosaic virus, KoMV) 屬於 Potyvirus 屬，是目前彩色海芋產區中導致病毒流行病的重要病原之一。遭受 KoMV 感染的海芋植株會出現嵌紋、綠島、褪綠、葉形扭曲、花梗縮短、花色斑駁等病徵，降低海芋的商品價值。本研究的第一個目的是探討蚜蟲傳播彩色海芋 KoMV 的傳播生態學；第二個研究目的是發展容易操作、快速、經濟、靈敏度高的病毒檢測法，用來偵測富含黏液植物的病毒感染。於蚜蟲傳播 KoMV 的研究中，共檢測六種蚜蟲傳播 KoMV 的能力。結果顯示，桃蚜 (Myzus persicae) 和棉蚜 (Aphis gossypii) 可以在實驗室的條件下傳播 KoMV。桃蚜和棉蚜在感染 KoMV 的葉片上取食 10 分鐘，然後轉移到健康海芋 24 小時，就能成功地傳播病毒，符合非持續性的病毒傳播模式。許多生物和非生物因素可能會影響病媒昆蟲傳播植物病毒。桃蚜和棉蚜生活史中不同的發育階段與形態，對蚜蟲傳播 KoMV 的傳播效率沒有顯著差異。環境溫度對這兩種蚜蟲傳播 KoMV 的效率也沒有影響。另外，本論文所研發的快速病毒檢測法，不僅可成功檢測感染彩色海芋的四種 potyviruses，也適用於檢測感染 KoMV 和芋頭嵌紋病毒 (Dasheen mosaic virus) 的彩色海芋與其他富含黏液的植物。值得一提的是新發展的病毒檢測法之偵測靈敏度高於酵素連結免疫吸附分析法(ELISA) 和以 TRIzol 試劑萃取 RNA 再進行反轉錄聚合酶連鎖反應(RT-PCR) 的方法。本研究不但提供蚜蟲傳播 KoMV 的傳播生態學相關資訊，並發展了快速且靈敏度高的病毒檢測方法，將有助於往後研擬彩色海芋 KoMV 的防治策略。Calla lilies (Zantedeschia spp.) are ornamental plants in the family Araceae and native to southern Africa. They were introduced into Taiwan for producing cut flowers and pot plants from New Zealand in 1990s. Their colorful bulbous flowers have won the affection of consumers. Nevertheless, bacterial soft rot and viral diseases are the major limiting factors for calla lily cultivation. Konjak mosaic virus (KoMV), belonging to genus Potyvirus, is one of the main virus causing epidemics in the calla lily-producing areas. The symptoms of KoMV-infected calla lilies are mosaic, green islands, vein chlorosis and distortion on leaves; short peduncle and discolored spots on flowers. This virus decreases the marketable value of calla lily. The first objective of this research was to study the transmission ecology of KoMV in calla lily by aphid vectors. The second objective was to develop a simple, rapid, inexpensive and sensitive sample preparation method for the detection of plant virus infection in mucilaginous plants by reverse transcription-polymerase chain reaction (RT-PCR). Six aphid species were examined for their abilities to transmit KoMV, and Myzus persicae and Aphis gossypii were able to transmit the virus in laboratory conditions. M. persicae and A. gossypii transmit KoMV with an acquisition access period of 10 min and an inoculation access period of 24 hours, which corresponds with the features of non-persistent transmission manner. There are many biotic and abiotic factors affecting the transmission of plant viruses by insect vectors. There was no significant difference in KoMV transmission efficiency by M. persicae and A. gossypii among developmental stages and morphs. The temperature during virus acquisition and inoculation has no effect on the transmission of KoMV by these two aphid species. For developing a rapid virus detection method of calla lily-infecting viruses, crude RNA extraction with the YG buffer following with RT-PCR assay not only successfully detected the infection of four potyviruses in calla lily but also was suitable for detecting KoMV and Dasheen mosaic virus in calla lily and other mucilaginous plants. Furthermore, this new developed protocol was demonstrated to be more sensitive than enzyme-linked immunosorbent assay (ELISA) and TRIzol RNA extraction following with RT-PCR assay. The knowledge of transmission ecology and the development of rapid and sensitive virus detection assay would be valuable for developing disease control strategies for KoMV in calla lily