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Detection of virus infected lima bean seed and their effects on plant production
|關鍵字:||lima bean;萊豆;seed;Seed-borne virus;CMV;BCMV;種子;種傳病毒;胡瓜嵌紋病毒;菜豆普通嵌紋病毒||出版社:||園藝學系所||引用:||王惠亮、張穎櫻、張清安。2005。花生條斑病毒Ts 系統基因序列之解讀與分析。植物病理學會刊14:211-220。 宋妤。2005。萊豆。臺灣農家要覽增修訂三版農作篇（二）。豐年社 p. 557-560。臺北。 邱人璋。1993。四十年來台灣植物病毒及擬菌質體病害的研究植物病毒與似病毒病害研討會專刊。行政院農業委員會編印。1-28頁。 張清安 1994 台灣花卉病毒病害。台灣花卉病蟲害研討會專刊中華植物保護學會編印277頁。213-224頁。 郭井泉。1992。控制大豆花葉病毒危害的種子帶毒率臨界水平。東北農學院學報23:220-225。 郭井泉、何平、張明厚、呂文清。1991。SMV種傳特性及其田間流行的種子傳毒率預測。病毒學雜誌6：171-178。 張清安。 2005。種傳病毒之特性、檢測與管理。植物病理學會刊14:77-88。 張清安。2008。作物健康種苗在病害管理上之回顧與展望。「節能減碳與作物病害管理」研討會。農試所、台中。 張清安、陳金枝、楊佐琦、詹竹明。2002。無病毒豇豆種子之研發、推廣與展望。植保會刊 11：107-111。 張清安、郭燕君。2001。豇豆病蟲害防治曆 P. 1-15。蔬菜病蟲害綜合防治專輯第五版。行政院農委會中部辦公室編印。 鄧汀欽。2007。種子健康驗證體系之種傳病毒的檢測。植物重要防疫檢疫病害診斷鑑定技術研習會專刊(六)：53-66。 廖吉彥、張清安、蔡錦慧、賴瑞聲、鄧汀欽。2004。感染通天草(Uraria crinita)的花生條斑病毒（Peanut stripe virus）之鑑定。植保會刊 46：379-390。 Adams, D. B. and C. W. Kuhn. 1977. Seed transmission of peanut mottle virus in peanut. Phytopathology 67:1126-1129. Albrechtsen, S. E. 2006. Testing methods for seed-transmitted viruses: Principles and Protocols. 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萊豆(Phaseolus limensis Macf.)為自交作物，目前尚無專業採種或健康種苗繁殖體系，農民栽培萊豆習慣自行留種，致使病毒有機會經由種子傳播到幼苗。於2008年6月至2009年11月期間分別於台中和台南共7處萊豆田採集疑病葉片，以10種豆類病毒抗體進行間接法免疫酵素分析(indirect ELISA)。結果在76個樣本中，與potyviruses抗體正反應的45個樣本當中，包括40個BCMV、3個BCMNV、42個BlCMV、25個BYMV、11個PSbMV、36個PStV及3個CMV，28個樣品具疑似病徵，卻無法從上述抗體反應中檢出，經調查萊豆病毒病主要由菜豆普通嵌紋病毒(Bean common mosaic virus, BCMV)及胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)所引起。將與BCMV抗體呈陽性反應之樣本，經單斑分離得到病毒分離株(c8, c9和c12)，以Pot I-HRP5引子進行RT-PCR，經核苷酸序列比對，結果c9與Accession U34972.1 (PStV)相同度最高為89.2%。另有CMV分離株(CMV-lima)以Cucumovirus-專屬引子:CPTALL-3及CPTALL-5進行RT-PCR，經核苷酸序列比對，顯示CMV-lima與subgroup IB(strains Nt9 and Tfn)相同度為95-96%。病毒鑑定結果指出BCMV為Peanut stripe strain，CMV為subgroup IB。2008年收集學甲地區8位農戶自留種及7家種子行之市售種子，以BCMV與CMV抗體進行indirect ELISA。結果農戶自留種80顆種子中測出帶病毒率為BCMV(12.5％)和CMV(41.3％)，市售種子105顆中帶病毒率為BCMV(3.8％)和CMV(4.8％)。取4家市售種子播種，540株萊豆苗中，有1株感染BCMV，1株感染CMV。單獨與複合接種BCMV或CMV的帶病毒株種子，進行種子種苗病毒檢測，部分種子種苗帶有病毒，但在後續觀察成株無病徵發生。萊豆植株感染BCMV或CMV後，病毒均會侵染至種子，但僅分佈於種皮，種皮的病毒大多於發芽時隨種皮脫落，BCMV或CMV經種子傳播感染幼苗的比率遠低於種子帶病毒(seed-born)的比率。矮性萊豆感染不同CMV strain 對其株高、側枝數、產量與豆莢畸型比率皆有顯著影響，當中又以CMV-Safflower危害最嚴重。使用無病毒萊豆種子或自留種子生產萊豆，於冬季調查豆莢單莢重量及種子重量，皆顯著比春季高，且感染病毒比率也比春季低，產量以自留種子產量較高。
Lima beans (Phaseolus limensis Macf.) are self-pollinating crops, and there is at present no professional seed and nursery production system for certificated disease-free seeds. Bean farmers often keep their own planting stocks, and therefore viruses may be transmitted from seeds to plants. In this study, suspected infected leaf samples of lima beans were collected from Taichung and Tainan from June 2008 to November 2009, and indirect ELISA was performed with 10 antibodies for the detection and differentiation of different bean viruses. In the 76 specimens tested, 45 exhibited a positive reaction to potyvirus antibodies, including 40 to BCMV, 3 to BCMNV, 42 to BlCMV, 25 to BYMV, 11 to PSbMV, 36 to PStV and 3 to CMV. In addition, in 28 specimens suspicious symptoms were observed, but viruses were not detected by the antibodies. Further analysis revealed that viral infection of the lima beans was caused in the main by the bean common mosaic virus (BCMV) and the cucumber mosaic virus (CMV). Virus isolates (c8, c9 and c12) were obtained through single lesion isolation from BCMV-positive specimens, and the isolates were subjected to RT-PCR with a Pot I-HRP5 primer pair and then sequenced. The results demonstrated that isolate c9 had a maximum identify of 89.2% with the sequence of Accession U34972.1 (PStV). CMV isolates (CMV-lima) were also prepared and analyzed by RT-PCR using Cucumovirus-specific primers CPTALL-3 and CPTALL-5. The sequencing results indicated a identify of up to 95-96% between the CMV-lima isolates and subgroup IB (strains Nt9 and Tfn). The virus identification results confirmed that the BCMV identified in the beans was a strain of the peanut stripe virus (PStV), and the CMV was of the subgroup IB. Seed samples from the planting stocks of 8 bean farmers and 7 commercial seed nurseries were obtained from the Syuejia Township area in 2008 and were analyzed by indirect ELISA with BCMV and CMV antibodies. The analysis showed the virus-carrying rates of the 80 seeds from famers' stocks to be 12.5% and 41.3% for BCMV and CMV, respectively, while the rates of the 105 seeds from seed nurseries were 3.8% and 4.8% for BCMV and CMV, respectively. Among 540 seedlings grown from seeds collected from four seed nurseries, one was infected with BCMV and one with CMV. When the seeds from single or multiple BCMV- or CMV-inoculated plants were analyzed, virus was detected in some of the seeds, while no disease symptoms were observed subsequently in their seedlings and plants. In BCMV- and CMV-infected plants, the virus was normally harbored in the seeds but only distributed in the seed coats. As seed coats were normally detached during germination, the virus did not stay with the seeds, and therefore the rate of transmission of BCMV or CMV to seedlings was far lower than the seed-born virus prevalence. Varied significant effects were observed in lima beans infected with different CMV strains, including reduction in plant height, lateral branch numbers, yield and pod malformation rates. Of the different CMV strains, CMV-Safflower was observed to have the worst effect. Comparing the use of virus-free lima seeds or the seeds from farms, the single pod weight and seed weight were significantly higher in the winter than the spring, the infection rate in winter was lower than that in the spring; and the yield was higher with the farmers'seeds than with the virus-free seeds.
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