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Title: | 洛神葵癌腫病之病原鑑定與特性分析 Identification and characterization of the causal agent of roselle crown gall disease |
Authors: | Huan-Yu Chen 陳奐宇 |
Advisor: | 林乃君(Nai-Chun Lin) |
Keyword: | 洛神葵,洛神葵癌腫病菌,抗菌敏感性試驗,整合害物防治管理, Roselle,Agrobacterium radiobacter,Antimicrobial susceptibility test,Integrated Pest Management, |
Publication Year : | 2018 |
Degree: | 碩士 |
Abstract: | 洛神葵在臺灣種植面積約為 124.88 公頃,其中又以臺東縣栽培面積最大,2016 年首次於臺東縣多良村與歷坵村洛神葵 (Roselle) Hibiscus sabdariffa Linn. 栽培田區的植株上發現異常腫瘤,隔年調查顯示該病害的發生地區有逐漸擴展之趨勢,涵蓋範圍在臺東知本以南,估計田間罹病率約為 0.6-10 %。採集病株並自腫瘤處分離到細菌菌株,該菌株於胰蛋白大豆瓊脂培養基 (Tryptone soy agar,TSA) 上呈乳白帶有粉紅色澤之黏稠菌落,經柯霍氏法則 (Koch's postulates),確認其對洛神葵具有病原性,能夠造成莖部產生腫瘤,且能自腫瘤處再被分離,應為此病害之病原菌。此菌能在胡蘿蔔切片試驗中形成腫瘤,於穿透式電子顯微鏡下確認其為多根周生鞭毛之桿狀菌,經由 16S rDNA 序列比對、recA 專一性引子對偵測及 Biolog 細菌鑑定系統鑑定,此病菌為 Agrobacterium radiobacter,其生理生化特性近似 Agrobacterium rubi。本病害在臺灣植物病害名彙尚無紀錄,屬於洛神葵之新病害,將此病害命名為洛神葵癌腫病 (Roselle crown gall disease)。洛神葵癌腫病菌於 20-37 °C 溫度之下均能生長,適合生長於 pH 6-7 中性偏酸之環境;在有製造傷口的情況下,莖部穿刺接種與土壤澆灌接種皆可於洛神葵植株上造成腫瘤,尤以接種於莖頂分生組織所造成之腫瘤顯著大於其他接種部位且會造成植株生長矮化。當以莖部穿刺接種法接種時,最低致病病原菌濃度為 104 cfu/mL,且在濃度為 109 cfu/mL 時所形成的腫瘤顯著大於其他較低濃度的組別。將剪枝器具浸泡於 75 % 酒精與 3 % 次氯酸鈉 (NaOCl) 溶液中 3 分鐘可有效降低經修剪造成感染的可能性。此外,在培養基上對此病菌進行潛力微生物與化學藥劑之篩選,其中又以 Pseudomonas protegens XH1-2a、Pantoea ananatis A63 與 Bacillus amyloliquefaciens PMB01 菌株以及四環黴素 (Tetracycline)、鏈四環黴素 (Streptomycin+Tetracycline) 與鋅錳乃浦 (Mancozeb) 有較佳生長抑制效果。於溫室防治試驗中,施用 P. protegens XH1-2a 雖然仍有腫瘤產生,但無論是保護處理或是同時處理均可有效降低腫瘤大小;利用四環黴素作為保護處理效果較佳,而同時處理則幾乎可以抑制腫瘤形成。受洛神葵癌腫病菌感染的罹病植株生長勢會變衰弱,雖不致造成死亡,但嚴重者亦會影響生長發育與產量。本研究鑑定出洛神葵癌腫病之病原,並進一步了解其特性,將有助於後續探討其發生生態與防治技術,以降低此病害所造成之損失。 In Taiwan, the cultivation area of roselle is about 124.88 hectare. Taitung county is main region for production of roselle. Since 2016, a new crown gall disease was found in the commercial plantations of roselle (Hibiscus sabdariffa) throughout Taimali and Jinfong in Taitung County. In summer 2017, we found the disease spread out to the other areas South of Ziben in Taitung County, and the estimated disease incidence was about 0.6-10%. Through Koch’s postulate, it was proved that the bacterial strains isolated from crown galls formed on roselle are pathogenic to roselle. They can induce gall formation on the stems of roselle, from which the same bacterial strain can be re-isolated, indicating that it should be the causal agent. Colony morphology of the isolated bacterial strain is viscous and milky white in color on the tryptone soy agar (TSA). In a carrot slice bioassay, tumor formation could be observed on the cambial regions after inoculation of the isolated strains. The pathogenic bacteria isolated in this study are rod-shaped and have peritrichous flagella. By means of 16S rDNA sequence analysis, recA-specific primers and Biolog microbial identification system, they were identified as Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942 (Synonyms: Agrobacterium tumefaciens and Rhizobium radiobacter) whose physiological and biochemical characterisitics are similar to Agrobacterium rubi. As this disease was not recorded in “List of Plant Diseases in Taiwan” and is a new disease of roselle, it was then named “roselle crown gall disease”. A. radiobacter can grow from 20 to 37 °C, and pH 6-7 is optimal for its growth. It can induce tumor formation by stem puncture inoculation or soil drenching as long as wounds are present. When stem puncture inoculation was used, the minimal infection dose is 104 cfu/mL. Furthermore, significantly bigger galls were formed on meristems when the inoculum was 109 cfu/mL. Disinfection of equipment with 75 % ethanol and 3 % NaOCl can decrease the possibility of infection through pruning. Furthermore, Pseudomonas protegens XH1-2a、Pantoea ananatis A63 and Bacillus amyloliquefaciens PMB01, as well as tetracycline, streptomycin+ tetracycline and mancozeb, can inhibit growth of A. radiobacter in a disk diffusion test. In a greenhouse assay, although tumors were still formed after application of P. protegens XH1-2a, smaller galls could be observed when plants were treated with P. protegens XH1-2a before or together with A. radiobacter. Application of tetracycline before pathogen inoculation could effectively suppress gall formation. Tumor was barely formed while roselle plants were inoculated with A. radiobacter mixed with tetracycline. A. radiobacter could weaken roselle plants. Although it would not kill roselle plants, A. radiobacter might affect growth and yield of roselle when the diseas is getting worse. In this study, the causing agent of roselle crown gall disease was successfully identified and characterized, which would help us further investigate its ecology and develop control strategies for prevention or treatment of roselle crown gall disease to reduce the economic loss. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72112 |
DOI: | 10.6342/NTU201803903 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 植物醫學碩士學位學程 |
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