兩種豇豆微斑駁病毒台灣分離株之分子與生物特性之探討

Analyses of molecular and biological characteristics of two isolates of Cowpea mild mottle virus from Taiwan

簡蘭懿(Lan-Yi Chien)

指導教授 ： 張清安 ； 鄭櫻慧

朝陽科技大學/理工學院/應用化學系生化科技碩博士班/碩士(2011年)

摘要

本研究於嘉義新港採集到疑似感染病毒的長豇豆及在南投草屯收集到葉片呈現嚴重斑駁甚至萎縮，植株矮化生長不良之菜豆，將此兩種樣品分別機械接種於奎藜葉片上經過三次單斑分離建立純系病毒分離株。採集由病葉抽取之總量RNA以carlavirus之簡併式廣效引子進行基因選殖與定序，所增幅的片段其序列與GenBank上的序列比對結果，發現這兩種分離株與GenBank上登錄為Cowpea mild mottle virus (CpMMV) 之分離株 (GU191840) 於鞘蛋白 (CP) 基因之核?酸及胺基酸序列相同度分別達89 % 及97.6 %，顯示兩者皆屬CpMMV之分離株。以下稱豇豆分離株為CpMMV-V，而菜豆分離株為CpMMV-P。此乃台灣地區豆科植物感染CpMMV之首次紀錄。利用電子顯微鏡陰染法於兩分離株病汁液中均可觀察到彎曲長絲狀，長度約為 600 nm的病毒顆粒。透過機械接種進行寄主範圍測試，多數豆科植物均可被成功感染，並發現兩分離株在寄主反應上有些許的差異。為取得CpMMV二分離株之全長基因體序列，以病毒之double-stranded RNA為模板，利用所設計之九組引子對，經過RT-PCR總共獲得九個相互重疊之增幅片段，經逐一套疊連接後分別獲得兩個全長為 8236 bp與 8243 bp之序列。分析兩序列均具有六個開放轉譯架構 (ORF) ，包括最上游可能具有複製?功能之ORF 1 ，其次依序為ORF 2、3及4 所組成之三基因區塊 (triple gene block) ，可能為病毒結構鞘蛋白之ORF 5 及最下游之ORF 6 。此種基因體架構與Carlavirus屬病毒特性亦完全吻合。比較此兩株分離株六個開放轉譯架構之核?酸與胺基酸序列相同度皆可達 73.0 - 88.8 % 與 74.8 - 97.9 % 以上，其中CP基因更高達 88.8 % 與 97.9 % 的相同度，印証兩者應屬同ㄧ病毒之不同分離株。此外，將兩序列與目前在GenBank上已登錄之 18 個CpMMV與Carlavirus屬病毒進行全長與鞘蛋白序列比對，發現以美國波多黎各所報導的GU191840與巴西落花生上所報導的DQ444266之分離株與本研究之兩分離株之相同度最為接近。利用PHYLIP軟體所分析繪製之親源關係演化樹，亦證實台灣兩個CpMMV分離株間之緊密親源性。本研究以飼育於聖誕紅並確認無帶毒之銀葉粉蝨 (Bemicia argentifolii bellows and perring) 進行傳毒試驗，發現其傳毒能力僅能維持一天。測試粉蝨於病株上吸食時間與獲毒之關係，發現只要吸食 10 分鐘即可獲傳毒能力，若吸食時間達到 1 hr其傳毒率可達最高。當固定粉蝨獲毒時間為 24 hr時，測試不同傳毒吸食所需時間，發現獲毒粉蝨只需在健株上吸食 10 分鐘就可達到 100 % 的傳毒率。上述試驗證實粉蝨可於極短的吸食時間下即可獲毒，且獲毒後不需潛伏期可立即傳毒，而傳毒能力只能維持一天，此等特性的確符合非永續型傳播 (non-persistent transmission) 之要件。此ㄧ特性與部份文獻對CpMMV之傳播特性之敘述吻合。本研究亦證實兩個CpMMV台灣分離株均無法經由病株種子帶毒傳播至後代幼苗。綜合以上分子與生物特性探討結果，可知本研究之兩分離株為台灣首度發現可透過粉蝨以非永續型方式傳播的豇豆微斑駁病毒，由於研究中証實其可廣泛感染國內多種重要經濟豆類作物，因此預估其具有對本省豆類作物生產造成威脅之可能性。

關鍵字

豇豆微斑駁病毒

並列摘要

A diseased plant of asparagus bean with mild foliar symptoms and samples of common beans showing severe virus symptoms were collected in 2009 in the fields of Chiayi and Nantou, respectively. By mechanical inoculating these samples onto Chenopodium quinoa, chlorotic spots were found to develop on the inoculated leaves of both isolates and single lesion pure isolates were established subsequently. Using total RNA extracted from C. quinoa infected leaves as template, expected DNA fragment was amplified in RT-PCR by a carlavirus specific degenerate primer pair and found to share 89 % and 97.6 % in the nucleotide and amino acid sequence identities, respectively with Cowpea mild mottle virus (CpMMV) (Acc. No. GU191840) after cloning and sequencing analyses. This result indicated that the two isolates from asparagus and common beans were both isolates of CpMMV and therefore they were designated hereafter as CpMMV-V and CpMMV-P, respectively. This is the first report of the occurrence of CpMMV in Taiwan. Electron microscopy studies confirmed that flexuous elongated virus particles about 600 nm in length were observed in the leaf dips of CpMMV-V and CpMMV-P. All the 12 legume species mechanically inoculated could be infected by these two CpMMV isolates, except that the pathogenecity of CpMMV-P to most of legume hosts was stronger than CpMMV-V. To obtain the full length genomic sequences of CpMMV-V and CpMMV-P, we use their viral dsRNA as templates and 9 designed primers for amplification in RT-PCR and subsequently obtained 9 different overlapped DNA fragments. After alignment analyses, full length genomic sequences of CpMMV-V and CpMMV-P were revealed to be 8236 bp and 8243 bp in length, respectively. Both their complete sequences encoded six open reading frames (ORFs) including the most upstream ORF 1 possibly having polymerase activity, ORF 2, 3, and 4, the triple gene block, ORF 5, the structural coat protein, and the ORF 6 with unknown function. This genome structure was identical to those of the members of Carlavirus. Comparing the 6 ORFs between CpMMV-V and CpMMV-P, 73 - 88.8 % and 74.8 - 97.9 % of nucleotide and amino acid identities were found. Expectedly, these two viruses shared the highest nucleotide and amino acid identities between their ORF 5, the CP of virus particle, indicating they were strains or isolates of the same virus species. When compared sequence identities of these two CpMMV isolates with 18 CpMMV isolates reported from other geographic regions, the Puerto Rico cowpea isolate (GU191840) and Brazil peanut isolate (DQ444266) were found to have closest sequence homology. By the use of PHYLIP software, two phylogenetic trees illustrating the genetic evolutionary relationship of CpMMV-V and CpMMV-P with 18 other CpMMV and with other known carlaviruses were plotted; both strongly showed high degree of homology between these two Taiwanese CpMMV isolates and with other reported CpMMVs. Different from most carlaviruses with aphid transmissibility, CpMMV was reported however by some workers that it was transmitted non-persistently by whiteflies. Our transmission experiment using silver leaf whiteflies (Bemicia argentifolii Bellows and Perring) rearing on Poinsettia as vectors also showed that CpMMV-V and CpMMV-P could be transmitted by whiteflies with non-persistent manner. Our results presented the evidence that whiteflies could efficiently acquire both isolates by feeding on diseased plants for as short as 10 minutes; without latent period viruliferous whiteflies could readily transmit the viruses as long as they were given 10 minutes of access feeding on healthy plants; and the transmissibility of viruliferous whiteflies could only maintain 1 day. These findings on the whitefly transmissibility clearly revealed that the two Taiwanese CpMMV isolates shared similar biological properties with other known CpMMV reported previously. Besides whitefly transmission, CpMMV was found by some workers that it could possibly be seed-borne. However, our studies using four different legume crops including mungbean, asparagus bean, adzuki bean and common bean as infected hosts, none of the seeds collected from these hosts showed CpMMV infection after germination. Based on the biological and molecular properties of these two CpMMV isolates presented herein clearly indicated that they shared similar characteristics of CpMMV reported in the literature. Due to this is the first report of the occurrence of CpMMV in Taiwan, its current distribution in different regions and possible effect on the production of legume crops should be alerted and evaluated.

並列關鍵字

Cowpea mild mottle virus

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