台灣梨樹病毒病害之鑑定及發展應用於田間調查和檢疫之檢測技術

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台灣梨樹病毒病害之鑑定及發展應用於田間調查和檢疫之檢測技術

Etiology of viral diseases of pear in Taiwan and development of detection methods for field survey and quarantine

吳榮彬 , Ph.D　　Advisor：詹富智　　

英文

蘋果莖凹陷病毒；蘋果黃化葉斑病毒；蘋果莖痘斑病毒；梨樹病毒病害；梨接穗；田間調查；檢疫； ASGV ； ACLSV ； ASPV ； pear disease ； pear scion ； disease damage ； quarantine

Abstract │ Reference (352) │ Altmetrics

Abstract 〈TOP〉

梨(Pyrus spp.)為台灣重要栽培的果樹作物之ㄧ。目前已知至少有6種薔薇科果樹病毒可以感染梨樹，其中造成蘋果及梨產業受到經濟危害，且普遍感染梨樹的重要病毒為蘋果莖凹陷病毒(Apple stem grooving capillovirus, ASGV)、蘋果黃化葉斑病毒(Apple chlorotic leaf spot trichovirus, ACLSV)及蘋果莖痘斑病毒(Apple stem pitting foveavirus, ASPV)。過去，台灣從未有任何梨樹病毒病害的相關報導，本研究目的即針對在台灣梨樹上發現的重要梨樹病毒，進行鑑定及特性分析，並發展快速的病毒檢測技術，以應用在病毒的田間調查及進口梨接穗檢疫上。為了了解重要的梨樹病毒是否已經存在於台灣的梨栽培產區，並且危害台灣梨樹，因此本研究初期即針對產生小葉、葉片叢生、褪綠黃化、斑駁及畸形葉等，疑似病毒病害病徵的罹病植株，以酵素連結抗體免疫吸附法(enzyme-linked immunosorbent assay, ELISA)及反轉錄聚合酶鏈鎖反應(reverse transcription-polymerase chain reaction, RT-PCR)進行偵測後，發現ASGV、ACLSV及ASPV已存在於梨樹罹病株中。但是，初期尚未進行病毒的鑑定，對其感染特性也不清楚。因此，再進一步針對在台灣梨樹上所發現的三種梨樹病毒，進行病毒分離、鑑定及特性分析。在本研究中，在田間發現一個從未報導過，且引起小葉及葉畸形病徵的梨樹病害，並且自罹病葉中分離出一長絲狀病毒分離株(TS1)後即著手進行鑑定。經鞘蛋白基因序列比對及特性分析後，確定此病毒分離株為ASGV。再利用病毒回接試驗(back-inoculation)，確認在田間所觀察到，引發小葉畸形的新病害-梨葉畸形病，即為ASGV所引起的。另以病毒鞘蛋白基因所進行的親源關係分析結果得知，本土ASGV病毒分離株與日本及韓國分離株間的親緣關係較為接近。此外，亦從帶有褪綠、黃化葉斑病徵的梨樹上分離出一長絲狀的病毒分離株(LTS1)，經序列比對及特性分析確定為一新的ACLSV分離株。藉由病毒回接試驗，確認ACLSV即引起田間所觀察到病徵的病原，並利用超薄切片的方式，觀察到ACLSV在梨樹細胞中累積分佈情形。再將37個不同來源ACLSV分離株的鞘蛋白胺基酸序列進行分析後，配合其親緣關係分屬結果，發現不同寄主及地區來源的ACLSV分離株，可區分成亞洲I、II、III及歐洲型等四種地理型，且其演化上的分屬，會依循地緣性的不同而有差異，但與分離株的寄主來源並無相關性。另外，我們再從帶有葉脈黃化病徵的橫山梨上，分離出另一種長絲狀病毒分離株(VY1)，經鑑定及序列分析後確定為ASPV病毒。再以病毒回接試驗，確認分離株VY1即為田間橫山梨上觀察到引起葉脈黃化病徵的病原。而經由序列比對後，發現可將本文中20個ASPV分離株，依照鞘蛋白基因長度的不同，可區分成三種型態，而且在鞘蛋白基因序列的5′端變異性較大。但相反的，在這些分離株的鞘蛋白基因序列的3′端，有660核苷酸(220胺基酸)的為高度保留區(conserved region)。將這些分離株間先以全長度鞘蛋白基因序列比對後，依彼此間序列相同度上的差異，區分成A、B、C和D等4群，再利用鞘蛋白基因3′端高度保留區，進行彼此間序列的比對後，發現各分離株間，核苷酸及胺基酸序列比對值(81.1-95.5% nt 及 91.8-99.1% aa)明顯高於利用全長鞘蛋白基因所比對者(74.8-91.2% nt 及 79.4-93.7% aa)，而這個發現將可提供一有利的方式，去進行ASPV種間的分類，並探討各分離株間的親緣關係。此外，為了解台灣田間病毒分佈及其危害影響情形，在2010年3到5月份間，針對疑似病毒病害病徵的葉片進行逢機採樣後，再利用本研究所發展的ELISA及RT-PCR技術進行病毒檢測，以了解這些樣本受到病毒感染的情形。並且利用所建立的梨病毒檢測技術，也已在2001到2010年間應用在進口梨接穗檢疫上，以偵測ASGV、ACLSV及ASPV三重要梨樹病毒。在受檢疫的3,782樣本中，有13個樣本被檢測出受ASGV單一感染，3個為ACLSV，1個ASPV及1個複合感染ASGV及ACLSV。總括以上，經本研究所完成的病毒鑑定及特性分析結果，確認ASGV、ACLSV及ASPV三種病毒分別為在台灣橫山梨樹上造成小葉畸形(leaf distortion)、黃化葉斑(chlorotic spot)及葉脈黃化(vein yellows)等病徵的病原。而在本研究中所發展的RT-PCR及ELISA偵測技術，也已應用在田間調查及進口梨接穗的檢疫上。

Parallel Abstract 〈TOP〉

Pear (Pyrus spp.) is cultivated worldwide and it is one of the important fruit crops in Taiwan. At least 6 rosaceous fruit tree viruses were reported to infect pear plants. Severe disease damage and yield loss were reported in apple and pear cultivations due to the infection by Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). No viral diseases were reported in Taiwan in the past several decades. The objectives of this study were to identify and characterize the major viral pear diseases in Taiwan and to develop rapid and reliable detection methods for pear viruses in field surveys and in imported pear scions for quarantine purpose. Pear plants (P. pyrifolia var. Hengshen) showing virus-like symptoms, such as reduction in size of foliage, tufted, leaf chlorosis, leaf mottling and leaf deformation, were found in the orchards in central Taiwan. The viruses of ASGV, ACLSV and ASPV were detected on these diseased pear plants by the enzyme-linked immunosorbent assay (ELISA) and the reverse transcription-polymerase chain reaction (RT-PCR). Sequentially, the identifications, characterizations and etiology of ASGV, ACLSV and ASPV were investigated. A pear disorder showing symptoms of reduced size of foliage and leaf distortion was never described previously. However, a virus culture (TS1) with filamentous virion was isolated and identified from the above-described symptomatic pear. Sequence analysis of the coat protein (CP) gene indicated that isolate TS1 belongs to ASGV. Back inoculation of pear seedlings with TS1 confirmed that ASGV was the causal agent of this new pear disease, named pear leaf distortion. The phylogenetic analyses of the ASGV CP genes revealed that the Taiwanese ASGV isolates were closely related to Japanese and Korean pear isolates. Another virus culture (LTS1) with filamentous particles was isolated from a disease pear plant showing chlorotic spots and ring pattern on the leaves. Sequence analysis and biological characterizations indicated that LTS1 is indeed an isolate of ACLSV. Pathogenicity of ACLSV isolate LTS1 was confirmed by back-inoculation to pear seedlings. Virus particles accumulated in the cytoplasm were observed in the ultrathin sections of LTS1-infected pear leaf tissues. Phylogenetic analysis revealed that the available 37 ACLSV isolates originated from various hosts and geographic locations could be grouped into four clusters, i.e. Asia I, II, III and Europe. Moreover, our study provides the phylogenetic relationships among ACLSV populations correlated with their geographic origins based on their amino acid sequences of CPs. Thirdly, a virus culture (VY1) with filamentous virion was isolated from a symptomatic pear leaf showing symptoms of vein yellows (VY). It was characterized as an isolate of ASPV by sequence analysis. Isolate VY1 was identified as the casual agent of the PVY disease on Hengshen pear by back inoculation. The CP genes of the present 20 ASPV isolates were grouped into three sub-types based on nucleotide sizes which showed high sequence variability at 5′-terminal portions. A conserved region of 220 amino acids was identified in the C terminal of the CP genes of these 20 ASPV isolates. They were further divided into four groups, A, B, C and D, on the basis of sequence comparisons. The sequence comparisons of the CP genes in the inter-groups showed 74.8-91.2% and 79.4-93.7% of nucleotide and amino acid identity, respectively. However, when the nucleotide and amino acid sequences of the 660 nt 3′-CP conserved regions were compared among the ASPV isolates, the nucleotide and amino acid sequence identity values rose to 81.1-95.5% and 91.8-99.1% respectively. This finding of using 3′-CP conserved regions for sequence identity comparison among ASPV isolates provides better demarcating criteria for the taxonomy of ASPV. In order to understand the pear virus distribution and their possible influence in pear cultivation in Taiwan, pear plants grown in eight major agricultural regions were surveyed for the related incidences of ASGV, ACLSV and ASPV from March to May of 2010. The most common virus-like symptoms observed in the fields included mottling, mosaic, leaf distortion, chlorosis, reduction in size of foliage, and deformation. Tests of symptomatic leaf samples randomly collected from pear orchards were conducted with the developed ELISA and RT-PCR protocols to verify the virus infections on pear plants. In addition, the ELISA and RT-PCR protocols were used for the detection of ASGV, ACLSV and ASPV in the pear scions bearing the flower buds imported from Japan from Oct 2001 to Jan 2010. Thirteen, three and one out of the 3,782 tested pear scions were detected to be infected with ASGV, ACLSV and ASPV, respectively. One out of these samples was detected as co-infected with ASGV and ACLSV. In summary, we had accomplished the identification and characterization of ASGV, ACLSV and ASPV that induced symptoms of leaf distortion, chlorotic spots and vein yellows, respectively, on domestic Hengshen pear cultivar in Taiwan. Furthermore, the ELISA and RT-PCR systems developed in this study are being routinely applied for field surveys of pear viruses and detections of viruses in imported pear scions for quarantine purpose.

Reference ( 352 ) 〈TOP〉

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