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研究生:梁專譯
研究生(外文):Neoh, Zhuan Yi
論文名稱:菲律賓葫蘆科Begomovirus病毒基因體歧異度及其地理分佈之探討
論文名稱(外文):Genetic Diversity and Geographic Distribution of Cucurbit-infecting Begomoviruses in the Philippines
指導教授:蔡文錫
指導教授(外文):Tsai, Wen-Shi
口試委員:陳金枝林志鴻蔡文錫
口試委員(外文):Chen, Chin-ChihLin, Chih-HungTsai, Wen-Shi
口試日期:2022-07-28
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:植物醫學系
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2022
畢業學年度:111
語文別:英文
論文頁數:54
中文關鍵詞:捲葉病南瓜捲葉菲律賓病毒、南瓜捲葉中國病毒番茄捲葉新德里病毒東南亞
外文關鍵詞:Leaf curl diseaseSquash leaf curl Philippines virusSquash leaf curl China virusTomato leaf curl New Delhi virusSoutheast Asia
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葫蘆科植物是全球重要的經濟作物,且使用於人類日常飲食。然而,由粉蝨傳播的Begomovirus所引起的瓜類捲葉病 (Cucurbit leaf curl disease, CuLCD) 限制了作物的生產,造成嚴重的產量損失 (可高達100%),尤其在熱帶和亞熱帶地區。目前瓜類捲葉病已發現於東南亞的印度尼西亞、馬來西亞、菲律賓、泰國和越南等地。造成東南亞瓜類捲葉病的Begomovirus主要有番茄捲葉新德里病毒 (Tomato leaf curl New Delhi virus, ToLCNDV)、南瓜捲葉中國病毒 (Squash leaf curl China virus, SLCCNV) 及南瓜捲葉菲律賓病毒 (Squash leaf curl Philippines virus, SLCuPV)。菲律賓於1977年首次報告有瓜類捲葉病之發生,隨後於2003年,在採自呂宋島Nueva Ecija的感病南瓜上,被鑑定出其病原為 SLCuPV (原名SLCCNV-[Philippines])。2006年則於呂宋島 Benguet的感病佛手瓜樣品中鑑定出SLCCNV。然而,菲律賓至今仍然缺乏廣泛性的瓜類捲葉病調查及其相關病毒資料。因此,本論文探討菲律賓瓜類捲葉病的Begomovirus病毒基因體歧異度及其地理分佈狀況。利用於2018至2019年間,採自菲律賓全國60個田區,具有嵌紋、捲葉、黃化、植物矮化等病徵,共249個葫蘆科的樣品。使用廣效引子對進行PCR檢測後,共有103個樣品測得Begomovirus。根據採集地點和年份,獲得38條SLCuPV DNA-A、6條SLCCNV DNA-A和42條SLCuPV DNA-B全長度基因體序列,經序列分析後,菲律賓的SLCuPV及SLCCNV 分離株分別可再分為A和B兩個株系。病毒專一性檢測顯示,SLCuPV是主要造成菲律賓瓜類捲葉病的Begomovirus,且在南瓜和扁蒲樣品中皆有檢測出SLCCNV與 SLCuPV複合感染的現象,但在這些複合感染樣品中都沒有檢測出SLCCNV DNA-B。此外,亦以選取的病毒分離株確認了病毒病原性。本研究結果提供菲律賓瓜類捲葉病Begomovirus病毒基因體歧異度及其分佈概況,可作為日後強化病害管理,特別是針對菲律賓和東南亞瓜類捲葉病的抗病選育提供重要參考基礎。
Cucurbits are important economic crops cultivated worldwide and contributed to the human daily diet. However, the cucurbit leaf curl disease (CuLCD), caused by whitefly-transmitted begomoviruses constrains crop production resulting in significant yield losses (up to 100%), especially in tropical and subtropical areas. In the Southeast Asia, CuLCD has been reported in Indonesia, Malaysia, the Philippines, Thailand and Vietnam. Meanwhile, three major begomoviruses, Tomato leaf curl New Delhi virus (ToLCNDV), Squash leaf curl China virus (SLCCNV) and Squash leaf curl Philippines virus (SLCuPV) were associated with CuLCD. In the Philippines, the CuLCD was firstly reported in 1977, and then the disease-associated SLCuPV (formerly named as SLCCNV-[Philippines]) was identified from squash in Nueva Ecija, Luzon in 2003. In 2006, SLCCNV was also identified from the diseased chayote in Benguet, Luzon. However, there is still weak on the destruction of CuLCD with limited virus information in the Philippines. Here, the genetic diversity and geographic distribution of CuLCD associated begomoviruses in the Philippines were conducted. During 2018 to 2019, 249 cucurbit samples with disease symptoms of mosaic, leaf curling, yellowing and plant stunting were collected from 60 locations throughout the Philippines. One hundred and three samples were detected positively for begomoviral DNAs by polymerase chain reaction with the virus detection primers. Based on locations and years, 38 full-length SLCuPV DNA-A, 6 full-length SLCCNV DNA-A and 42 full-length SLCuPV DNA-B sequences were obtained. Consequently, SLCuPV and SLCCNV Philippines isolates can be grouped into 2 strains, respectively. Virus specific detections revealed that SLCuPV was the major begomovirus associated with CuLCD in the Philippines. Mixed infection of SLCCNV with SLCuPV was detected in pumpkin and bottle gourd samples. Interestingly, no SLCCNV DNA-B was detected in the samples. Furthermore, the pathogenicity of selected virus isolates was confirmed. The research provides virus genetic diversity associated with CuLCD for strengthening the disease management, especially in developing resistant cultivars against CuLCD in the Philippines as well as in the Southeast Asia.
中文摘要 I
Abstract II
誌謝 III
Context IV
Introduction 1
Molecular characteristics and taxonomy of begomoviruses 1
CuLCD in the Southeast Asia 3
Cucurbit-infecting begomoviruses in the Philippines 5
Management of CuLCD 6
Objective of research 7
Materials and Methods 8
Sources of CuLCD samples 8
Extraction of viral DNAs 8
Detection of begomovirus 9
Cloning of begomoviral DNAs 9
Partial sequence analysis of begomoviral DNAs 12
Amplification and cloning of full-length begomoviral DNAs 12
Sequence analysis of full-length begomoviral DNAs 13
Specific detection of begomoviral DNAs 13
Construction of begomoviral infectious DNAs 14
Agrobacteria transformation 15
Agroinoculation of host plants 16
Results 17
Begomovirus detection 17
Begomovirus DNA sequence analysis 17
Specific detection of cucurbit-infecting begomoviruses in the Philippines 19
Pathogenicity of major cucurbit-infecting begomoviruses in the Philippines 19
Discussion 21
References 25
Tables 34
Table 1 Begomovirus detection of cucurbit samples collected in the Philippines. 34
Table 2 Sequences of primers used in this study. 35
Table 3 Sequences of begomoviral DNAs used in this study. 37
Table 4 Sequence characteristics of cucurbit-infecting begomovirus DNAs identified in this study. 38
Table 5 Nucleotide sequence identity (%) among DNA-A sequences of cucurbit-infecting begomoviruses in the Philippines. 40
Table 6 Nucleotide sequence identity (%) among DNA-B sequences of cucurbit-infecting begomoviruses in the Philippines. 41
Table 7 Specific detection of begomoviral DNAs in cucurbit samples collected in 2018-2019 in the Philippines. 42
Table 8 Pathogenicity of SLCuPV infectious clones from the Philippines. 43
Table 9 Pathogenicity of SLCuPV and SLCCNV infectious DNA-As from the Philippines. 44
Figures 45
Figure 1 Phylogenic tree obtained from the alignment of complete DNA-A and DNA-B nucleotide sequences of cucurbit-infecting begomoviruses. 45
Figure 2 1.2% agarose gel electrophoresis of specific detection of cucurbit-infecting begomoviruses in the Philippines. 46
Figure 3 Diagram of constructed infectious DNAs of SLCuPV-A[PH-BoG137-18] and -A[PH-Pk71-19]. 47
Figure 4 Diagram of constructed infectious DNAs of SLCuPV-A[PH-Pk212-41-19]. 48
Figure 5 Diagram of constructed infectious DNAs of SLCuPV-B[PH-Pk76-18] and -B[PH-WM114-18]. 49
Figure 6 Diagram of constructed infectious DNAs of SLCuPV-B[PH-BoG216-18]. 50
Figure 7 Diagram of constructed infectious DNAs of SLCCNV-A[PH-Pk195-18], -A[PH-Pk11-19] and -A[PH-Pk212-42-19]. The viral ORFs are shown and labelled by arrows. 51
Figure 8 Symptoms on agroinoculated host plants with infectious DNAs of SLCuPV strain A. 52
Figure 9 Symptoms on agroinoculated host plants with infectious DNAs of SLCuPV strain B. 53
Figure 10 Symptomless tobacco (Nicotiana benthamiana) plants after agroinoculation with infectious DNA-As of SLCuPV and SLCCNV. 54
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