Pierce’s disease (PD) of grapevines caused by a xylem-limited bacterium, Xylella fastidiosa (Wells, 1987) is one of the plant diseases under international quarantine regulation. The formation of bacterial biofilms, gums and tyloses occlude xylem vessel and then restrict xylem flow. The exhibited symptoms of PD in the grapevines include leaf marginal necrosis, chlorosis, and matchstick petiole. This insect-borne bacterial disease and its vectors have been investigated since 2002 in central Taiwan. Bothrogonia ferruginea (Fabricius, 1787) was considered as a candidate vector based on the positive detection for X. fastidiosa by PCR from field-collected insects. In this study, the basic ecological and biological information for B. ferruginea, including field surveys and life history parameters, and transmission efficiency of X. fastidiosa to grapevines were studied. The field survey data demonstrated that the B. ferruginea population peak occurred in April and May in Miaoli County and from July to August in Nantou County. The developmental time of B. ferruginea reared on pilose beggarticks (Bidens pilosa var. radiata) at 25oC from egg to fifth instar nymph, were 8.4, 8.2, 9.2, 15.4, 20.4, and 18.5 d, respectively. The adult longevity of female and male was 36.1 ± 9.6 and 32.3 ± 6.0 d, respectively. Forty two to seventy percentage of B. ferruginea acquired X. fastidiosa from field-collected infected grapevines within 24 h acquisition assess period in grouping vectors trial. Inoculation efficiency for grouped and individual vector trials was 18.2 and 6.7%, respectively. Insect-inoculated plants did not show typical symptoms of PD until 4 months post-inoculation and X. fastidiosa could be isolated from infected plants. Successful acquisition and transmission of X. fastidiosa to healthy grapevines by B. ferruginea suggesting this leafhopper was the insect vector for the transmission of the PD to grapevines. Although the population density and transmission rate of B. ferruginea suggested that it has the low possibility in triggering PD epidemic, the adult longevity and high consumption rate resulted in its high acquisition and retention ability. Additionally, in view of the body size as well as strong flight ability might imply that this vector plays an important role in reservoir and dissemination for X. fastidiosa. Therefore, a right-timing control of this vector should not be ignored. According to the data from field survey and life history of B. ferruginea, the control action against PD vector should be taken from March to August around epidemic area in Miaoli County and Taichung City, Taiwan. The management of this vector should pay attention to weeding around vineyard, especially pilose beggarticks, an appropriate host plant of B. ferruginea. In further study, we will aim at the microbial control with entomopathogenic fungi against this vector to suppress the occurrence of PD.葡萄皮爾斯病 (Pierce’s disease, PD) 屬於國際檢疫病害，由葡萄皮爾斯病病原菌 (Xylella fastidiosa (Wells, 1987)) 引起，此種細菌侷限在寄主植物導管內增殖、產生嚴重阻塞、引起植物水分運輸受阻而導致缺水現象，使病株呈現葉緣焦枯、黃化及提早落葉的病徵。自 2002 年起陸續在臺灣監測罹病葡萄株及其媒介昆蟲，經由田間監測與分子檢測結果，可自野外採集之黑尾大葉蟬 (Bothrogonia ferruginea (Fabricius, 1787)) 蟲體內偵測到 PD 病原之特定分子序列，將黑尾大葉蟬列為候選媒介昆蟲。本研究即探討黑尾大葉蟬之生態資料、生物特性及其媒介 X. fastidiosa 之傳播效率。黑尾大葉蟬於田間發生盛期，在平地為四至五月，而在中海拔地區則為七至八月。於室內 25oC 定溫條件下，以大花咸豐草 (Bidens pilosa var. radiate) 飼育黑尾大葉蟬，其卵期至五齡若蟲發育時間依序為 8.4、 8.2、 9.2、 15.4、 20.4 及 18.5 天，雌成蟲平均壽命為 36.1 ± 9.6 天，而雄成蟲平均壽命為 32.3 ± 6.0 天。黑尾大葉蟬進行 24 小時之獲菌試驗，其獲菌率高達 42~70%。傳播試驗分別以單隻及五隻一組進行，其傳播效率為 6.7 及18.2％。經媒介昆蟲接種的植株需等待四個月之潛伏期，才表現葡萄皮爾斯病病徵，並可從接種植株內再分離出相同的病原菌，故黑尾大葉蟬能成功自罹病葡萄植株獲菌並傳菌至健康葡萄植株上，證實黑尾大葉蟬具有傳播 X. fastidiosa 之能力，確立其在臺灣葡萄皮爾斯病媒介昆蟲的地位。雖黑尾大葉蟬的野外族群密度及傳播率較低，但因成蟲壽命及高取食率能使黑尾大葉蟬有較高的獲菌率及保菌能力，且該葉蟬的體型大又具有良好的飛行能力能，將有助於病原菌的散播及保存，因此不可忽視其在病媒防治上的重要性。根據田間調查和生活史資料，顯示大花咸豐草是黑尾大葉蟬的寄主植物，該葉蟬之防治應於每年三至八月期間，加強在苗栗及台中疫區的葡萄園區外圍之雜草管理，以杜絕其滋生或傳播病原的機會。未來研究將著重在微生物防治上，利用蟲生真菌防治黑尾大葉蟬，壓制其在野外族群的密度，以減少葡萄皮爾斯病之發生。Contents 摘要 i Abstract iii Contents v Figure of Contents vii Table of Contents ix Introduction 1 Materials and Methods 4 1. Insect Collecting and Rearing 4 2. Grapevine Plant Culture 5 3. Isolation and Culture of Xylella fastidiosa 5 4. Detection of Xylella fastidiosa in Insect Body and Leaf Petiole by Polymerase Chain Reaction (PCR) 6 4.1 DNA Extraction from Insect Body 6 4.2 DNA Extraction from Leaf Petiole 8 4.3 PCR Assay 9 4.4 Detection Threshold of Xylella fastidiosa in Insect Body and Leaf Petiole by PCR 10 5. Mechanical Inoculation 10 6. Life History Study 11 7. Morphometric Data 12 8. Transmission of Xylella fastidiosa to Grapevines by Bothrogonia ferruginea 12 8.1 Transmission Test Conducted by Grouping of 5 Adults 12 8.2 Transmission Test Conducted by Single Adult 13 9. Statistical Analysis 14 Results 15 1. Monitoring of Population Density of Bothrogonia ferruginea 15 2. Life History Parameters 16 3. Morphometric Data 17 4. Completion of Koch’s Postulates 18 5. Transmission Efficiency 18 5.1 Grouping in 5 Adults 18 5.2 Single Adult 21 Discussion 22 References 29 Figures and Tables 37 Appendix 5
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