Identification of Squash leaf curl Philippines virus on Benincasa hispida in Taiwan

Abstract

During a disease survey of wax gourd (Benincasa hispida Cogn.) in Taiwan, virus-like symptoms of yellowing, vein enation, rugose mosaic and leaf curling were observed and further characterized. A naturally infected wax gourd sample (Wg1) was collected from experimental field at Beidou, Changhua County, Central Taiwan. The plant was tested negative with indirect ELISA using the antisera against eight RNA viruses commonly found in cucurbits, including Cucurbit aphid-borne yellows virus, Cucumber green mottle mosaic virus, Cucumber mosaic virus, Melon vein-banding mosaic virus, Papaya ringspot virus-watermelon type, Watermelon silver mottle virus and Zucchini yellow mosaic virus. However, it showed positive to a geminivirus, tentatively named isolate of Squash leaf curl virus Wg1 (SqLCV-Wg1), with polymerase chain reaction (PCR) using primer pair that is specific to begomovirus genomes. After cloning and sequencing, the PCR product was identified to be 1116 nucleotides (nt) in length, encompassing 126, 771, and 219 nt of the AV2, CP, and AC3 gene coding regions, respectively. Sequence alignment with the corresponding regions of other begomoviruses revealed that SqLCV-Wg1 is most closely related to Squash leaf curl Philippines virus, with percent nucleotide identities of 97.6, 97.7, and 93.8 for AV1, CP, and AC3 genes, respectively. The CP gene of SqLV-Wg1 was further cloned into the vector pET21d(+), which was then used to express proteins in Escherichia coli BL21(DE3) and to produce specific antiserum for future diagnosis purposes. This is the first record of cucurbitaceous plants infected with Squash leaf curl virus in Taiwan. 冬瓜(Benincasa hispida Cogn.)上發現一種新病害病徵，葉部褪綠、葉脈突起及褪色、葉面皺縮、甚至捲葉之現象，使得植株生長勢衰弱。採自彰化縣北斗鎮之冬瓜疑似病毒罹病株(Wg1)，經瓜類作物的八種病毒血清檢測，包括瓜類蚜媒黃化病毒(Cucurbit aphid-borne yellows virus, CABYV)，胡瓜綠斑嵌紋病毒(Cucumber green mottle mosaic virus, CGMMV)，胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)，甜瓜脈綠嵌紋病毒(Melon vein-banding mosaic virus, MVbMV)，木瓜輪點病毒-西瓜系統(Papaya ringspot virus W type, PRSV-W)，番茄斑點萎凋病毒（(Tomato spotted wilt virus, TSWV)，西瓜銀斑病毒(Watermelon silver mottle virus, WSMoV)，及矮南瓜黃化嵌紋病毒(Zucchini yellow mosaic virus, ZYMV)，均早陰性反應。針對begomovirus外鞘蛋白之高度保留區域核酸序列，經比對設計引子對，以PCR增幅之。經由勝任細胞的轉形作用與核酸解序，獲得116個核甘酸，分為AV2 (126個核苷酸)、CP (771個核苷酸)及AC3 (219個核苷酸)。經與其他begomoviruses比對，得知Wgl病毒分離株與Squash leaf curl Philippines virus最有親源關係，其核酸相同度在AV1, CP, and AC3基因分別為97.6, 97.7, and 93.8%。利用載體pET21d (+)夾帶SqLV-Wg1的CP基因，在Escherichia coli BL21 (DE3)中表現病毒鞘蛋白，並用以注射紐西蘭白兔製備專一性的抗血清，供進一步診斷鑑定用。本文為台灣瓜類感染Squash leaf curl virus的首次紀錄。