玉米褪綠斑駁病毒（Maize chlorotic mottle virus）之親緣分析與台灣之發生調查

Phylogenetic Analysis and Occurrence of Maize chlorotic mottle virus Infecting Maize in Taiwan

周建銘(Chien-Ming Chou) ； 陳君弢(Chun-Tao Chen) ； 簡伊萱(Yi-Hsuan Chien) ； 蔡錦慧(Chin-Hui Tsai) ； 陳金枝(Chin-Chih Chen) ； 林鳳琪(Feng-Chyi Lin) ； 陳怡如(Yi-Ju Chen) ； 鄧汀欽(Ting-Ching Deng)

《台灣農業研究》 67卷2期 (2018/06) Pp. 191-208

https://doi.org/10.6156/JTAR.201806_67(2).0005

摘要

自雲林縣虎尾地區採集具褪綠、黃化及褐化乾枯病徵之玉米病害樣本，並將其機械接種於甜玉米'Honey-236'可產生類似褪綠斑駁病徵，並獲得一個分離株(稱為NFU2)。樣本以簡併式引子對進行反轉錄聚合酶鏈鎖反應(reverse transcriptase polymerase chain reaction; RT-PCR)，可增幅出預期大小(1.4 kb)產物，經解序後序列與玉米褪綠斑駁病毒(Maize chlorotic mottle virus; MCMV)中國Yunnan3分離株(Acc. No. JQ982469)相同度達99%。將MCMV NFU2分離株全基因體解序後，其全長為4,436 nt，經親緣分析後MCMV共可分為美國分離株、中國分離株、東非分離株及台灣分離株等四個分群；若將其鞘蛋白序列進行親緣分析，則可發現泰國之MCMV分離株應為近年於亞非大發生MCMV之共同起源。依解序所得MCMV NFU2分離株之鞘蛋白序列，構築於表現載體後大量表現以製備其多元抗血清，該多元抗血清經測試可用於田間樣本之酵素免疫分析法(enzyme-linked immunosorbent assay; ELISA)檢測。此外，利用其鞘蛋白序列設計專一性引子對MCMV-g3514F/MCMV-g4014R，可以RT-PCR於單隻玉米薊馬中檢測出MCMV，可作為後續媒介昆蟲傳播MCMV的機制研究之用。於2014-2015年間利用MCMV及甘蔗嵌紋病毒(Sugarcane mosaic virus; SCMV)抗體於台灣各地玉米栽培區調查，共檢測306個樣本，結果顯示MCMV罹病率達79.7%，MCMV與SCMV複合感染造成之玉米致死性壞疽(maize lethal necrosis; MLN)罹病率為31.7%；檢測樣本中，白玉米、糯玉米、青割玉米、硬質玉米及7種甜玉米品種皆可檢出MCMV，食用玉米中「華珍」之罹病率較低，但亦達60%，青割玉米及硬質玉米MCMV罹病率平均達31.6%。本研究為台灣MCMV分離株之基因體全長度解序、親緣分析、檢測試劑開發及病害調查之首次報告。

關鍵字

玉米褪綠斑駁病毒 ； 甘蔗嵌紋病毒 ； 親緣分析 ； 檢測試劑 ； 病害調查

並列摘要

Maize samples showing chlorotic, yellowing and leaf necrosis symptoms were collected in Huwei Township, Yunlin County in February 2014. After inoculation by leaf sap, the similar chlorotic mottle symptom was developed on sweet corn cv. 'Honey-236' and a pure virus isolate ＂NFU2＂ was obtained from infected sweet corn by a serial inoculation. The total RNA of ＂NFU2＂ isolate was extracted to conduct a reverse transcriptase polymerase chain reaction (RT-PCR) with the degenerate primer (Tenui-DF1/Tenui-DR1) and that yields a 1.4 kb product. The product could combine into one single contig, and this contig shared 99% sequence identities with Chinese Maize chlorotic mottle virus (MCMV) ＂Yunnan3＂ (Acc. No. JQ982469). The complete sequence of MCMV ＂NFU2＂ was as 4,436 nucleotides. Through whole genome sequence alignment and phylogenetic analysis revealed that MCMV could be divided into four groups, including American isolates, Chinese isolates, East African isolates and Taiwanese isolate. Phylogenetic analysis with nucleotide sequences of MCMV coat protein showed that MCMV isolates which were prevalent in Asia and Africa in recent years could possibly share the common ancestor of MCMV from Thailand. MCMV coat protein sequence was constructed into expression system of Escherichia coli Rosetta strain and the expressed viral coat protein was purified for antiserum production. Homemade MCMV antiserum could be used in enzyme-linked immunosorbent assay (ELISA) for MCMV detection in field samples. Furthermore, based on MCMV coat protein gene, a specific primer pair (MCMV-g3514F/MCMV-g4014R) for RT-PCR was developed, and by which the MCMV could be detected from a single maize thrip (Frankliniella williamsi Hood). These detection tools are available for further study of insect transmission mechanism of MCMV. Survey of 306 maize samples in Taiwan from 2014 to 2015 by indirect ELISA using antibodies against MCMV and/or Sugarcane mosaic virus (SCMV). The results indicated that incidence of MCMV reached 79.7% and maize lethal necrosis (MLN) caused by MCMV and SCMV reached 31.7%. Maize samples including sweet corns, white corns, waxy corns, forage corns and dent corns were all MCMV-infected. Sweet corn cv. 'Bright Jean' has the lower incidence (60%) among the green corn and comparing with incidence 31.6% of forage corn and dent corn. This is the first report about complete genome sequence, phylogenetic analysis, detection reagent development and field survey of MCMV infecting maize in Taiwan.

並列關鍵字

Maize chlorotic mottle virus ； Sugarcane mosaic virus ； Phylogenetic analysis ； Detection reagent ； Field survey