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研究生:蔡米皓
研究生(外文):Mi-Hau Tsai
論文名稱:火鶴花細菌性葉枯病菌之分離、偵測與防治
論文名稱(外文):Isolation, Detection and Management of Xanthomonas axonopodis pv. dieffenbachiae Causal Organism of Bacterial Blight of Anthurium
指導教授:謝式坢鈺謝式坢鈺引用關係
指導教授(外文):S. P. Y. Hsieh
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系
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:2001
畢業學年度:89
語文別:英文
論文頁數:111
中文關鍵詞:火鶴花細菌性葉枯病菌火鶴花偵測防治枯草桿菌
外文關鍵詞:Xanthomonas axonopodis pv. dieffenbachiaeanthuriumDetectionManagementBacillus spp.
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火鶴花細菌性葉枯病菌之分離、偵測與防治
總摘要
台灣火鶴花栽培面積約123公頃,主要產地在中南部地區,1991年首次在南投縣種植的橘色系火鶴花上發現葉片上有黃暈壞疽型病徵之細菌性葉枯病(bacterial blight of anthurium),其後於1992年在南部種植的粉紅花系火鶴花上亦發現黃化型病徵,此後病害即遍及台灣的火鶴花產區,尤其在溫暖潮濕的氣候下,葉枯病發生相當嚴重,造成農民重大損失。本研究主要將各地所分離之菌株進行初步鑑定,以了解病原菌之特性,並利用隨機引子(random primer)對病原菌篩選之專一性片段,應用Polymerase Chain Reaction (PCR)技術發展出一套快速、靈敏和專一性之葉枯病菌檢測系統。另一方面篩選火鶴花葉表之拮抗細菌,以進行生物防治之試驗。分離到之83株葉枯病菌,以自后里地區所分離之XadH-2菌株致病力較強,發病之最低接種濃度為104cfu/ml。病原菌在葉片上之族群消長調查,結果顯示其在接種後第15天前已侵入組織中,故可作為防治時間上之參考。由罹病組織分離之細菌經生理生化性質測定及接種試驗,確定造成臺灣火鶴花葉枯病之病原細菌為Xanthomonas axonopodis pv. dieffenbachiae (Xad)。將Xad-2之genomic DNA及其對照菌株與17組隨機引子進行RAPD分析後,只有隨機引子OPB-1可對Xad genomic DNA增幅出一約565bp專一性片段。將此片段經TOPO cloning system選殖後,進行片段解序,再將此序列利用DNA star system設計其PCR正反向引子對,經合成後與所有Xad菌株進行Bio-PCR偵測,均能增幅出預期之341bp。此專一性引子對應用於火鶴花葉上進行Bio-PCR敏感性測定,其可偵測最低濃度為約4.6×104cfu/cm2的菌量。所篩選出來較佳之拮抗菌株No.2、3、23、33、36應用於火鶴花細菌性葉枯病上均有防治效果,第120天的葉枯病菌感染火鶴葉面積之級數分析,以No.2、3、23、33、36、BS002與枯萎寧、嘉賜銅、克枯爛等三種藥劑的效果較佳。在藥劑試驗上以嘉賜銅和銅鋅錳乃浦的防治效果較好,第120天時菌感染火鶴葉面積之級數分析,以處理銅鋅錳乃浦、嘉賜銅、鋅錳乃浦和克枯爛的防治效果較好。供試拮抗菌經鑑定為No.2和36為Bacillus amyloloquefaciens,No.23為Bacillus licheniformis,No.3和33為Bacillus subtilis。
Isolation, Detection and Control of Xanthomonas axonopodis pv. dieffenbachiae Causal Organism of Bacterial Blight of Anthurium
ABSTRACT
At present anthurium cultivation area is about 97ha in Taiwan. Bacterial blight disease on anthurium showing leaf symptoms of necrotic lesions that surrounded by yellow halo were first found at Puli, Nantou in 1991, and a leaf yellow type of symptoms of the disease was observed in the southern part of Taiwan in the following year. The disease is now spread to most of anthurium fields in Taiwan. In this study, efforts were paid on, identification, detection and control of the disease. Selected primers were used to find specific DNA markers of Xanthomonas axonopodis pv. dieffenbachiae (Xad) by using random amplified polymorphic DNA (RAPD). Antagonistic bacteria isolated from leaf surface were used to test for their efficacy on biocontrol. Isolate XadH-2 is the most virulent among 83 strains tested. The lowest concentration needed to cause infection was 104cfu/ml. The pathogen infected into leaf tissue 15th day after spray inoculation. Based on physiological, biochemical and inoculation test, the caused organism was identified as X. axonopodis pv. dieffenbachiae that cause anthurium blight. RAPD analysis for genomic DNA of Xad-2 and comparison with strains and 17 pairs of random primer, it was fount only OPB-1 could amplify a distinct fragment of 565bp. The inserted DNA fragment was digested by EcoR 1. The primers were designed and synthesized by the sequence of the inserted fragment. Detection of all Xad strains all were able to amplify a distinct fragment of 341bp by Bio-PCR. Sensitive test of primer pairs by Bio-PCR, that lowest detection concentration is about 4.6×104cfu/cm2. Antagonistic bacterium No.2, 3, 23, 33 and 36 have potential to control anthurium bacterial blight. By grading analysis of infected leaf area 120 days after test, that No.2, 3, 23, 33, 36, BS002 and three pesticides. Kasugamycin+ copper oxychloride and maneb+ coppersulphate gave good control of the disease. Maneb+ coppersulphate, Kasugamycin+ copper oxychloride, mancozeb and tecloftalam showed better control when using grade analysis of pathogen infected leaf area on 120th day. Using Biolog antagonistic that bacterium No.2 and 36 were identified as Bacillus amyloloquefaciens, No.23 was Bacillus licheniformis and No.3 and 33 were Bacillus subtilis.
目錄
總摘要--------------------------------------------------------------------------------------------1
英文摘要-----------------------------------------------------------------------------------------2
第一章、前人研究-----------------------------------------------------------------------------3
一、病害史(病原的發現)-----------------------------------------------------------------3
二、台灣火鶴花細菌性葉枯病之病害史--------------------------------------------------4
三、血清偵測及Biolog鑑定----------------------------------------------------------------5
四、病原菌對藥劑之感受性-----------------------------------------------------------------6
五、火鶴花細菌性葉枯病菌之偵測--------------------------------------------------------6
六、火鶴花細菌性葉枯病菌之生物防治--------------------------------------------------7
七、聚合酵素連鎖反應在植物病害診斷上之應用--------------------------------------7
八、引用文獻-----------------------------------------------------------------------------------9
第二章、火鶴花細菌性葉枯病菌之分離、鑑定及族群消長
壹、中文摘要-------------------------------------------------------------------------------- --14
貳、英文摘要------------------------------------------------------------------------- ---------15
參、前言---------------------------------------------------------------------------------- ------16
肆、材料與方法-------------------------------------------------------------------------- -----18
伍、結果----------------------------------------------------------------------------------- -----26
陸、討論----------------------------------------------------------------------------------- -----32
柒、引用文獻------------------------------------------------------------------------------ ----35
捌、圖表------------------------------------------------------------------------------------ ----37
玖、附錄------------------------------------------------------------------------------------ ----47
第三章、火鶴花細菌性葉枯病之防治
壹、中文摘要-------------------------------------------------------------------------------- --50
貳、英文摘要-------------------------------------------------------------------------------- --51
參、前言---------------------------------------------------------------------------------- ------52
肆、材料與方法------------------------------------------------------------------------------ -54
伍、結果---------------------------------------------------------------------------------- ------60
陸、討論---------------------------------------------------------------------------------- ------63
柒、引用文獻------------------------------------------------------------------------------ ----66
捌、圖表---------------------------------------------------------------------------------- ------68
玖、附錄---------------------------------------------------------------------------------- ------77
第四章、火鶴花細菌性葉枯病菌之專一性片段的篩選與病原菌之偵測
壹、中文摘要-------------------------------------------------------------------------------- --78
貳、英文摘要-------------------------------------------------------------------------------- --79
參、前言---------------------------------------------------------------------------------- ------80
肆、材料與方法------------------------------------------------------------------------------ -82
伍、結果---------------------------------------------------------------------------------- ------91
陸、討論---------------------------------------------------------------------------------- ------95
柒、引用文獻------------------------------------------------------------------------------ ----97
捌、圖表---------------------------------------------------------------------------------- ----100
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