Detection and incidence of Cactus virus X on pitaya in Taiwan

紅龍果(Hylocereus undatus Britt. & Rose)為近年自國外引進而逐漸風行全台之醬肉果樹，目前只有報告仙人掌病毒X (Cactus virus X, CVX)可以感染紅龍果。本研究調查台灣各地及金門地區紅龍果園，發現有疑似病毒感染病徵之植株分佈頗為普遍，依病徵型態可歸納為退綠斑點型、斑駁型、壞疽型及黃化型等四型。其中以斑駁型發生較廣二木研究由宜蘭所獲得之斑駁型紅龍果摧病株上經單斑分離獲得一病毒分離株(EL1)，以機械接種方式ELI可感染雞冠花造成系統性黃化斑點，於千日紅、藜、奎藜及紅藜則產生局部壞疽病斑。電子顯微鏡觀察ELI病毒顆粒為長絲狀，人小約490-510 13 nm。將ELI繁殖於奎藜，利用硫酸絕等密度平衡離心可獲得純化之病毒顆粒，並用以作為免疫用抗原製備出專一性抗血清。以電泳分析(SDS-PAGE)純化之病毒顆粒，其鞘蛋白基木單位（sllbunit Proteill）分子量約為30 kDa。所製備之抗血清於西方轉漬、SDS-免疫擴散反應及間接型酵素連接抗體法等免疫分析反應中均與其同源抗原產生明顯反應，而不與健康對照抗原反應。為釐清ELI與CVX之關係，根據已知之CVX鞘蛋白核甘酸序列設計引子對，利用反轉錄聚合酵素（RT-PCR）可由ELI組織所抽取之全量RNA中增幅出預期之1.2kbp之DNA片段。此片段經選殖與解序後證實屬於cVX3' 端基因體序列，包括部分oRFZ、ORF3、ORF4及ORF5 (coat protein)。取其中部分ORF4及完整ORF5片段共864個核甘酸（對應288個氨基酸，Access No. AY241392）與CVX (Access No. AF308158)相比對，分析發現ELI鞘蛋白基因之核甘酸序列與CVX相同度達95.3%，而兩者氨基酸相似度為92.8％。經上述各項特性之比對，證實ELI係CVX之一種系統。依摧病株不同部位及不同病徵組織，以間接法酵素連接免疫分析(ELISA)偵測，得知新生側芽及初期病徵（退綠斑點及斑駁），可測得較高ELISA反應值。經各地調查及採樣偵測，屏束縣紅龍果園CVX發生率約90％以上，金門縣約50%，其餘地區約60-70％。紅龍果品種保存園中CVX普查結果，國姓黃皮(A13)等20個品種植株仍未被CVX所感染，標示為健康母本，進而加以隔離，以供日後發展健康種苗繁殖體系之用。 Pitaya (Hylocereus undatus) plants showing viral symptoms of chlorotic spots, mottling, necrosis and yellowing were found in orchards around Taiwan. From diseased pitaya plant which a virus isolate was obtained through series of single lesion isolation. By host range test, electron microscope observation, immunosorbent electron microscopy, coat protein electrophoresis, Western blotting, and RNA sequencing, the virus was identified as an isolate of Cactus virus X (CVX). The virus systemically infected Celosia argentea and caused chlorotic spots, but locally infected Gomphrena globosa, Chenopodium amaranticolor, C. quinoa, C. murale caused necrotic lesions. Electron microscopy of diseased tissue showed filamentous virus particles with 490-510 13 nm. The virus was purified from inoculated leaves of C. quinoa by isopycnic centrifugation in cesium sulfate and injected in rabbit for antiserum production. In SDS-PAGE, the virus was found consisting of one species of coat protein subunit and its molecular weight was estimated to be 30 kDa. In SDS-immunodiffusion, indirect ELISA and Western blot, the antiserum was found to specifically with its homologous antigen. Total of 864 nucleotides from the 3'-end region of this virus were sequenced and compared with the Cactus virus X from in GenBank (Access No. AF308158) showing the nucleotide identity index of the whole CP to be 95.3% , and the amino acid similarity index to be 92.8%. Using indirect ELISA to detect the CVX on different tissues of pitaya revealed that virus titers are higher in young shoots or parts with initial symptoms (chlorotic spots and/or mottling) than any other tissues tested. The incidence of CVX occurrence on pitaya orchard was surveyed around the Taiwan area and was found to be above 90% in Pintung , 50% in Kinman and from 60-70% in other areas. To find the CVX-free materials, a census was conducted to examine all the plants preserved in pitaya variety collection orchard located in National Pingtung University of Science and Technology. From this orchard a total of 77 samples were indexed out of which only 20 pitaya plants of 20 varieties were considered to be healthy. All of the healthy plants were separately multiplied for setting up the healthy pitaya seedling propagation system

Studies on a CMV Isolate From of Spinach Yellow Mosaic Disease

緒言 前人研究 材料與方法 甲、材料 一、試驗植物來源 二、育苗準備 乙、方法 一、汁液接種與單斑分離 二、不同離心力病毒之分離與接種試驗 三、還原劑對本病毒病原性之影響 四、病毒之純化與汁液接種試驗 五、物理性質測定 六、罹病材料超薄切片與病毒在組織內分佈 七、蛋白鞘分子量大小之電泳動測定 八、抗血清製備與抗體IgG 抽取 九、力價測定及純度測定 結果 一、汁液接種後寄主反應與單斑分離 二、不同離心力之分離試驗 三、還原劑對病原性之影響 四、病毒之純化與電子顯微鏡觀察 五、物理性質測定 六、超薄切片與病毒在組織內分佈 七、蛋白鞘分子量之電泳動測定 八、抗血清製備與抗體IgG 抽取 九、力價測定與純度判

感染冬瓜的南瓜捲葉病毒之鑑定

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

Molecular characterization of economically important viruses infecting fruit and energy crop in Taiwan

台灣向日葵葉片退綠斑點病，其葉片粗汁液接種奎藜會形成局部性病斑，經三次單斑分離後得到一個病毒分離株（SF-1）。經電子顯微鏡陰染法觀察其外型病原為長絲狀病毒，大小約770 x 13 nm，罹病組織超薄切片可觀察到細胞質束狀及風車狀圓柱內含體。以奎藜接種葉為材料進行病毒純化，經硫酸銫等密度平衡離心，所得病毒試料以膠體電泳分析測定鞘蛋白之分子量，估計為35kDa。以純化之病毒注射白兔製備抗血清，結果以瓊脂擴散反應測定，其抗血清只與同源反應。再經西方轉漬反應及間接法免疫酵素分析證實其血清有專一性，但本病毒以Agdia 之potyvirus group test kit 檢測亦呈正反應，因而將本病毒暫時命名為向日葵退綠斑點病毒(Sunflower chlorotic spot virus, SCSV)。抽取SCSV-SF-1全長的potyvirus核酸，將之定序並分析。 SCSV-SF-1核酸長度為9,741個核苷酸，可以轉譯成potyviruses的保留基本模組3,071胺基酸的polyprotein。 序列比較顯示3’端SCSV-SF-1與Bidens mottle virus (BiMoV)有96%相同性。 然而，SCSV-SF-1有狹窄的寄主範圍，不包括BiMoV的診斷寄主種類（鬼針草屬Bidens pilosa.和百日菊屬 Zinnia elegans）。 所以SCSV-SF-1是BiMoV一個分離株。 這是感染臺灣向日葵病毒病原BiMoV全長核苷酸序列的第一個報告。 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），均呈陰性反應。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DNA產物之選殖與核酸解序，獲得1116個核苷酸，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Wg1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SqLCV-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 Philippines virus 的首次紀錄。 洋香瓜等瓜類作物極具經濟價值，然近年來迭遭數種新興病毒病害之大規模感染，造成農民的重大損失。其中由菸草粉蝨(Bemisia tabaci species complex)所傳播的南瓜捲葉病毒Squash leaf curl Philippines virus (SqLCV，雙生病毒科)於台灣中、南部地區洋香瓜上造成捲葉病的肆虐。在2008下半年造成洋香瓜捲葉病害的大流行，甚而導致全園廢耕的嚴重經濟損失，成為洋香瓜產業的主要限制因子之一。因此急需開發方便有效的方法，以監測SqLCV於粉蝨與作物中的帶毒與感染狀況，預作適當之防治工作。本研究研發利用滾環式擴增法(Rolling Circle Amplification，RCA) 檢測雙生病毒環狀DNA的技術，持續於雲林、嘉義、與台南等縣市之主要瓜類作物栽培區進行SqLCV之監測。發現SqLCV普遍存在於田間瓜類作物與粉蝨族群中，但其基因體變異性極大。且帶毒率與感染率隨作物種類而不同，其中以西瓜最低，洋香瓜最高。為進一步了解雙生病毒基因體在不同時空環境中的變異性，本研究分析比對自2007年至2010年不同時間點，於台灣中南部分屬不同縣市行政區的地理位置洋香瓜專業栽培區所採集的SqLCV基因體序列，並分別比對來自於洋香瓜葉片或粉蝨體內所選殖之SqLCV基因體序列，發現在不同時空環境下經常發生變異的核苷酸位置較常出現於基因間隙區域(intergenic region)，而其他基因體區域相對較為穩定。不同的地理環境與瓜類作物對於雙生病毒的基因體序列亦有影響，因此基因體序列之變異性亦可作為追蹤雙生病毒來源之用。A disease of sunflower (Helianthus annuus) showing symptoms of chlorotic spot ,enation, yellow of leaves and stunted was collected from Puli, and from which a pure line of virus isolate (SF-1) was obtained through series of single lesion isolation. Before correctly identify the pathogen, A filamentous potyvirus about 770 x 13 nm in size was isolated from the infected plant and proved to be the causal agent of the disease. The molecular size of the coat protein of the pathogen was found to 35 kDa. The pathogen induced cylindrical inclusion body including the shapes of scrolls、laminated aggregated and bundles in host cytoplasm. According to preliminary test including westing blotting test、ELISA test、nucleic acid sequencing and amino acid sequencing, the 3’end region and analysis of identical relationship suggests this sunflower virus could belong to PVY subgroup and the name of “sunflower chlorotic spot virus”(SCSV) is tentatively porposed. The full-length genome of a potyvirus, previously known as sunflower chlorotic spot virus isolate SF-1 (SCSV-SF-1) which causes novel symptoms on sunflowers was sequenced and analyzed. The genome of SCSV-SF-1 is 9,741 nucleotides long, encoding a polyprotein of 3,071 amino acids containing the consensus motifs of potyviruses. Sequence comparison revealed that the 3’-terminus of SCSV-SF-1 shared over 96% similarities with isolates of Bidens mottle virus (BiMoV). However, SCSV-SF-1 has a very narrow host range, excluding the diagnostic host species for BiMoV, Bidens pilosa and Zinnia elegans. Therefore, SCSV-SF-1 is a distinct isolate of BiMoV. This is the first report of the full-length nucleotide sequence of BiMoV infecting sunflower in Taiwan. 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 Philippines virus in Taiwan. Cucurbits are economically important crops in Taiwan. However, several emerging viral diseases have devastated the production of cucurbits and caused great losses in recent years. Squash leaf curl Philippines virus (SqLCV, Geus Begomovirus , Family Geminiviridae), transmitted by whiteflies (Bemisia tabaci species complex) is among the most severe viral pathogens in cucurbits. The development of efficient and convenient methods for the monitoring of SqLCV in cucurbits is in need. In this study, modified rolling circle amplification (RCA) method to detect the circular DNA genome of SqLCV was developed and applied in the survey of SqLCV in whiteflies and cucurbit crops in the major production areas in southern and central Taiwan. The results revealed the common and persistent presence of SqLCV among various cucurbit crops and whitefly populations. The disease incidences and viruliferous ratios varied widely among different crops and whitefly populations, with watermelon and muskmelon being the lowest and highest, respectively. The spatial and temporal effects on the variations of SqLCV genomic sequences were further analyzed using samples collected from various localities belonged to different territorial district in Taiwan over the period of 2007 to 2010. Most genomic variations were identified in intergenic region sequences, while the others coding sequences were highly conserved. These results provide applicable references for the tracing of origins of SqLCV isolates and the design of effective disease management measures for SqLCV.摘 要 i ABSTRACT iii Literature review 1 Background: 1 Source of genetic variations of viruses 1 Significance of genetic variations of viruses 2 The scope of genetic variations investigated in this study 3 A. Economical significance of viral diseases 3 A1. Bidens mottle virus (BiMoV): 3 B. Taxonomy: 5 C. Genetic variations and evolutions 7 C1. Genetic variations in RNA viruses (potyviruses): 8 C2. Genetic variations in DNA virus (geminiviruses): 9 The Objectives of This Study 11 References: 12 Part I 19 Full-length sequence analysis of a distinct isolate of Bidens mottle virus infecting sunflower in Taiwan 19 Abstract 20 Introduction 21 Provenance of virus material 22 Sequence properties 22 Discussion 23 Reference 25 Table 26 Fig 27 Part II 32 Identification of Squash leaf curl Philippines virus on Benincasa hispida in Taiwan 32 Abstract 33 Introduction 34 MATERIALS AND METHODS 35 Field observation and virus source 35 Polymerase chain reaction (PCR) 35 Cloning and sequence analyses 35 Expression of coat protein (CP) gene 36 Preparation of CP-enriched fractions 36 Antiserum production 36 Indirect enzyme-linked immunosorbent assay(Indirect ELISA) 36 Result 37 Field observation. 37 Host range and ELISA test. 37 Polymerase chain reaction(PCR). 37 Sequence analysis. 38 Expression and purification of Wg1-CP gene. 38 Indirect ELISA. 38 Discussion 38 Reference 40 Table 1. 42 Fig. 43 Part III 49 Spatial and Temporal Analysis on the Genetic Diversity of Squash leaf curl virus in Muskmelons and Whiteflies in Central and Southern Taiwan 49 Abstract 50 Introduction 51 Materials and methods 53 Cucurbit leaf and whitefly samples 53 Rolling circle amplification (RCA) 53 Southern blot hybridization 53 DNA sequence analysis of viral genome 53 Results and discussion 54 Detection of geminivirus DNA by RCA 54 Southern blot hybridization to confirm the RCA method of DNA amplification 55 The surveys on SqLCV disease incidences in cucurbits and viruliferous ratios of whitefly populations by using RCA method 56 The analysis of Genetic relationship of SqLCV genome 57 Analysis of variability of SqLCV genome 57 Conclusion and prospectives 59 References 61 Table 64 Fig 6

Studies on spinach yellow mosaic disease. I. Isolation and identification of cucumber mosaic virus

平地菠菜的栽培，普遍發生黃化嵌紋病，將罹病植株經不同離心力分離，所得沉澱再分別用 Sepharose CL-4B 分子篩孔色層進一步純化，經汁液接種及電子顯微鏡觀察，發現在100,000 g 分離純 化試料之第一吸收峰處出現對菠菜有病原性，並觀察到大小約29 nm 的多量球形粒子存在。在汁液 接種測試中，對原寄主菠菜苗所出現的病徵與原來的症狀較相同，而認為是本病的主要病原。由單一 分離系(PL) 所得之病原，其物理性質為耐溫度性在50 - 55℃，耐稀釋性為1 0- 2 - 10- 3 倍，耐保存性在 4℃ 下時則為6 - 8天、室溫下為3 - 5 天。病毒鞘蛋白經S D S - PAGE 電泳其次單位蛋白分子量為2 5 K d。經家兔免疫注射製作血清其力價為1024 倍，以Ouchterlony 法分析血清類緣關係，甜椒C M V 與田間菠菜罹病汁液與純化病毒呈完成同質反應(homologus) 之現象。綜合試驗結果得知造成田間菠 菜黃化嵌紋病毒主要的病毒為胡瓜嵌紋病毒(Cucumber mosaic virus) 的一種。 Spinach grown in lowland areas of Taiwan was found widely exhibiting yellow mosaic symptoms, which is possibly caused by virus infection. A virus isolate (PL) was obtained from the symptomatic spinach and subsequently tested for its infectivity on 27 plant species from 9 Families by mechanical inoculation. Twelve species in Chenopodiaceae and Solanaceae but none of the plants from Leguminosae and Cucurbitaceae were infected. Inoculation of the PL isolate on spinach seedling induced symptoms similar to those observed from the fields. Infectious virus particles of PL were obtained by centrifuging the homogenates of the diseased tissue at 100,000 g for 60 min followed by chromatographing through Sepharose CL-4B column. In electron microscopy, the morphology of virus particles was found to be isometric about 29 nm in diameter. Similar sized and shaped virus particles were also detected in ultra-thin sectioned diseased spinach tissue. The thermal inactivation point of PL isolate is 50-55℃, dilution end point is 10- 2 to 10- 3 and the longevity in vitro is 3 to 5 days under room temp and 6 to 8 days at 4℃. By electrophoresis analyses, molecular weight of the coat protein subunit of PL was estimated as 25 kDa. An antiserum was prepared against the purified virions and found to react strongly with its homologous antigen but not to the healthy control spinach in SDS-immunodiffusion test. Furthermore, this antiserum gave indistinguishable reaction against antigens of PL isolate and an isolate of cucumber mosaic virus (CMV) from sweet pepper, indicating that PL isolate from spinach was serologically identical to CMV. This result corresponds to those documented in USA and Japan that CMV is a causal agent of spinach mosaic disease

Constructing a Knowledge Integrated Platform for High-Quality Crop

作物栽培管理決策必須綜合品種、土壤、氣象、地理、生態等動態即時性的整合資訊，才能實現高產、優質、高效的生產目標。國內農業試驗所目前已建置有稻作、土壤、氣象、病蟲害等資料庫或查詢系統，但分散各地，由各領域專家自行維護管理，尚未整合成單一窗口之服務系統。為建構國內農業生產整合服務體系，本計畫結合農業試驗所和各區農業改良場的不同領域專家，形成研發團隊，共同合作，擬將國內重要產業作物之栽培各面向知識進行研究及整合。計畫執行的重點，除整合國內現有各農業資訊系統外，同時也調查收集作物在不同地區之所有生長階段的生產動態性資訊，以增補知識整合平台所需的資料。透過本研究所建立的作物生產知識整合平台，能讓作物生產者和推廣輔導員都能快速、便利且正確地獲取品種選擇、生產知識、合理化施肥、病蟲害診斷防治、氣象災害預警防範等的整合型優質資訊。 Farming decisions require integrated knowledge of dynamic information related to variety, soil, climate, geography and ecology for achieving high yield, good quality, and high efficiency of crop production. In Agricultural Research Institute (ARI), various databases or query systems in rice, soil, climate, disease/pest have been developed and maintained by different specialists; however, they have not yet been integrated into a single-window service system. The object of this project is to construct an integrated service system of agricultural production for integrating knowledge of different specialties in crop cultivation for Taiwan's important crops. Our research team is composed of specialists who are working in various agricultural sectors such as ARI and all DARESs (District Agricultural Research and Extension Station). We will not only integrate existing agricultural systems, but also collect the dynamic information of crop production at all growing stages in different zones for fulfilling the data requests of integrated platform. This integrated knowledge platform can provide farmers and extensionists with quick and easy access to integrated information of variety selction, production knowledge, suitable fertilizer, disease/pest diagnosis as well as climate-damage prediction and early warning

Identification of Tobacco mosaic virus infecting Rehmannia glutinosa

從埔里地黃(Rehmannia glutinosa Libosch.)栽種區及本所中藥材栽培區採集各種疑似病毒感染的植株，汁液接種經單斑分離得到一純系病毒株(RG-1)。從寄主範圍試驗、電子顯微鏡觀察、凝膠雙擴散反應、電泳分析、西方轉漬反應及核酸與胺基酸序列比較，證實所分離的病毒株RG-1係屬於菸草嵌紋病毒(Tobacco mosaic virus, TMV)的一個獨立系統(strain)。本病毒接種奎藜(Chenopodium quinoa)後會產生局部壞疽病斑。經陰染法電子顯微鏡觀察病毒顆拉為長桿狀，大小約300-310 16-18 nm。病毒經接種至奎華繁殖，再利用硫酸銫等密度平衡離心法純化，純化所得試料為抗原用以注射白兔製備抗血清。電泳分析純化的病毒鞘蛋白，其主要蛋白分子量約為19 kDa。經西方轉漬反應、SDS-瓊脂免疫擴散反應(immunodiffusion)，自製的抗體與RG-1有同源(homologous)血清反應。從本病毒RNA 3'端解序480個核菅酸，與GenBank中的TMV (Access No. AY555269)比較鞘蚩白基因之核苷酸，其相同度達97.1%，而其胺基酸相似度則為99.4%。本報告為台彎地區TMV感染地黃植株的首篇紀錄。 Rehmannia glutinosa being a kind of medicinal plant cultivated in Taiwan was found to be infected by viruses. A plant with leaf mosaic was collected from Puli, and from which a pure line of virus isolate (RG1) was obtained through series of single lesion isolation. By host range test, electron microscope observation, immunosorbent electron microscopy, Ouchterlony immunodiffusion, coat protein electrophoresis, western blot analysis, and nucleotide sequencing, and analyses, the virus was identified as a isolate of Tobacco mosaic virus (TMV). The virus systemically infects Rehmannia glutinosa and causes mosaic symptom, but locally infects Chenopodium amaranticolor, C. quinoa and Nicotiana tabacum cv. Vam-Hicks and caused necrotic lesions. Direct negative staining by electron microscopy observation of diseased tissue revealed the presence of rod-shaped virus particles, around 300-310 16-18 nm. The virus was purified from inoculated leaves of C. quinoa by isopycnic centrifugation in cesium sulfate and the purified preparation was used to inject rabbit for antiserum production. The virus was found consisting of one species of coat protein subunit with an estimated molecular weight of 19 kDa. In Ouchterlony immunodiffusion and western blot analyses, the prepared antiserum reacted with homologous TMV antigens. The 3 - terminal 1044 nucleotides of this virus were cloned, sequenced and compared with corresponding sequences of representative Tobacco mosaic virus strains listed in public databases. It was found that this virus is most closely related to the Tobacco mosaic virus reported in China (Accession No. AY555269), with nucleotide and amino acid sequence identities of 97.1 % and 99.4 %, respectively. This is the first report of Tobacco mosaic virus infecting Rehmannia glutinosa in Taiwan

Investigation of the virus diseases on pepper in Taiwan

番椒（Capsicum annuum L., C. frutescens L.）主要分為甜椒和辣椒，為重要的茄科(Solanaceae)作物，台灣危害番椒病毒有11 種之多，自96 年起調查病毒在田間發生情形，甜椒樣本採集自露天栽培甜椒者，病毒發生率不高，各病毒檢出率差異不大，介於6.0-10.5%之間。設施栽培者，potyvirus 的檢出率遠高於其他病毒，且出現病毒病徵植株常呈現區塊存在，疑似人為傳播所致。檢測的辣椒樣本中，除了potyviruses，CMV 感染率最高，複合感染的比例高達80.5%。地球暖化雨量分布不均，很容易造成小型昆蟲大量發生，尤其是媒介病毒傳播的薊馬和粉蝨。98 年在仁愛鄉採集205 個番椒樣本，測到薊馬傳播的TSWV 感染比例達86.8%，99 年則降至32.0%。調查中粉蝨傳播的TYLCV 在番椒的感染率不高，鑒於其對番茄造成的嚴重危害，未來對番椒生產的影響亦須密切注意。 Pepper (Capsicum annuum L., C. frutescens L.) is divided into sweet pepper and chili pepper, as an important vegetable crops. There are 11 species of viruses infecting pepper in Taiwan. Pepper samples showed symptoms could be caused by virus were collected for analysis with ELISA and polymerase chain reaction (PCR). The virus infection rate of sweet pepper collected from open field is insignificant, ranging from 6.0-10.5% respectively. The infection rate of potyviruses in sweet pepper is much higher than other viruses if samples collected from net house. In chili pepper, the infection rate of CMV is the highest and 80.5% of samples showed co-infection with potyviruses. Since abnormal rainfall distribution of global warming, the population of virus vector may be increased and caused serious diseases. A severe disease on pepper plants showing chlorosis, necrosis and die back were observed in Renai Township in 2009. The causal agent is TSWV, a thrip transmitted tospovirus. The infection rate is higher than 30% but dropped to lower than 5% till 2010. In this survey, TYLCV, the whitefly transmitted virus does not show high infection rate in pepper. Since TYLCV caused dramatic losses in tomato, its influence in pepper production should pay more attention

Surveillance and Integrated Diseases and Pests Management of Ornamental Plants

本計畫調查景觀作物主要病蟲害種類及其週年消長，全年監測有無列名檢疫有害生物之發生情形，並測試主要病蟲害之防治藥劑效果並研發主要的非農藥防治技術。建立重要景觀作物有害生物相，建立防治病蟲害之環境管理規範及非疫生產點技術之研發。 This project focus on the study of management of insect pests and diseases of Ornamental plantsall year around. Some quarantine pests listed were surveyed and monitored. Development of the recommended measures and schedules and nonchemical control of pests and diseases were urgent requirement. The standard operation processes for pests and diseases control will be built up