孫岩章臺灣大學：植物病理與微生物學研究所吳魁偉Wu, Kuei-WeiKuei-WeiWu2007-11-272018-06-292007-11-272018-06-292007http://ntur.lib.ntu.edu.tw//handle/246246/58082迷迭香是為香草業者中重要的經濟作物之一，通常在夏天高溫多濕的環境下，易造成根腐病（Root rot），導致地上部黃化、枯死，經由鏡檢迷迭香根部組織，可在根部組織中發現卵孢子（Oospore）或厚膜孢子（Chlamydospore）等構造，初步懷疑係由卵菌綱（Oomycete）所引起，於是以選擇性培養基進行迷迭香根的病健部組織分離，所分離到的約120株分離株中，經初步鑑定出 Pythium 及 Phytophthora，且以 Pythium 之分離檢出率達98 ％ 為最主要，Phytophthora 之分離檢出率為2 ％。將所有120分離株依外部型態特徵做初步的鍵定與分群，共得20群，再逐一鑑定出其中有 Pythium 6種， Phytophthora 2種，此8種經分別接種於試管中已發根迷迭香扦插苗及穴盤介質栽培扦插苗，以檢定其病原性，結果發現，其中5種 Pythium 及2種 Phytophthora 皆具病原性，且兩類中分別以 Pythium myriotylum 及 Phytophthora nicotianae 最為主要。在藥劑防治上，以浸泡迷迭香盆栽地下部根系的方式，各施予500X鋅錳乃浦及500X甲基多保淨，浸泡時間分別為10秒、20秒及60秒，結果顯示，在浸泡鋅錳乃浦20秒與甲基多保淨10秒，皆有良好的防治效果。而以滴管澆灌此兩種農藥的方式，以檢定藥害情形，亦發現藥害情況並不明顯。在迷迭香所分離到的較強病原性的菌株中，選定腐霉菌 P. myriotylum 、 P. ostracodes 、 P. catenulatum 及疫病菌 Ph. nicotianae ，分別接種於甘藍、番茄和包心芥菜等3種蔬菜幼苗作物，其結果皆顯示可造成此3種蔬菜之根腐病徵。而在本研究中，已經由柯霍氏準則的驗證，證明迷迭香根腐病之發生、病原性和真菌種類，此對迷迭香根腐病的了解及後續之防治將有所幫助。Rosemary is one of the economically important fragrant herbs in agricultural industry. In warm and wet summer in Taiwan, however, rosemary is frequently affected with a root rot disease. The disease symptoms include leaf yellowing, wilt, and root browning. Under a microscope, we can find a lot of oospores and chlamydospores in the root tissue. Therefore it was thought to be caused by fungi of the family of Oomycetes. Selective medium developed by Ko et al for isolation of Pythium and Phytophthora was then used to isolate the suspect pathogen from browning roots from about 60 diseased plants. A total of 120 isolates were obtained from these 60 diseased rosemary plants, and among them 98 ％ were Pythium , only 2 ％ were Phytophthora . These 120 isolates were tentatively divided into 20 groups according to their morphology and other characteristics. Results showed that these isolates consisted of 6 species of Pythium and 2 species of Phytophthora . Among them 5 species of Pythium and both Phytophthora were pathogenic to roots of seedlings in test tubes and in pots, or inoculated to healthy pot seedlings. Pythium myriotylum and Phytophthora nicotianae were the dominant species, for these respective genus. The fungicides mancozeb and thiophanate methyl each at 500X concentration were applied by dip every the whole diseased plants for 10, 20 or 60 seconds. Results showed that mancozeb dipping for 20 seconds and thiophanate methyl dipping for 10 seconds are satisfactory for curing the disease. There two fungicides were not phytotoxicity to rosemary. Four root rot pathogens of rosemary including Py. myriotylum, Py. oedochilum, Py. catenulatum, and Ph. nicotianae were also used to inoculate healthy seedlings of cabbage, tomato, and mustard. They all showed root-browning symptom, after the inoculation. In conclusion, the Koch`s postulates of this disease has been proven, and the pathogens were identified. It should benefit the diagnose and control the disease in the future.內容目錄 口試委員會審定書…………………………………………………………….……..i 誌謝……………………………………………………………………………….…..ii 內容目錄………………………………………………………………………...……1 中文摘要……………………………………………………………………….… ….7 英文摘要……………………………………………………………………….……..8 第一章 前言…………………………………………………………………….….10 一、迷迭香的介紹………………………………………………………….….10 (一) 直立型迷迭香……………………….....................................................10 (二) 匍匐型迷迭香………………………………………………………... .11 二、迷迭香的栽培管理…………………………………………………..……11 三、迷迭香栽培上的問題………………………………………………….…..12 四、研究目的……………………………………………………………….…..12 第二章 前人研究………………………………………………………...………...13 第三章 材料與方法…………………………………………………………….….16 一、田間及盆栽迷迭香病害的調查…………………………………………...16 二、迷迭香根腐病病原菌株之分離、鑑定與培養……………………………18 三、供試土壤及介質之消毒與除菌……………………………………………20 四、迷迭香健康苗之繁殖………………………………………………………20 五、根腐病分離株對迷迭香之病原性檢定……………………………………20 （一）供試迷迭香健康株之準備……………………………….…………20 （二）供試 Pythium 與 Phytophthora 之游走子的製備…….……….…20 （三）病原性檢定方法………………………………………………….....21 1. 在試管中以無性繁殖扦插苗進行之病原性檢定……………...21 2. 以穴盤苗進行之病原性檢定…………………………………...21 （四）接種後植株根部的再分離………………………………………..22 六、迷迭香根腐病菌之藥劑防治試驗…………………………….………….22 七、迷迭香根腐病菌對三種蔬菜幼苗的接種試驗………………. ................23 第四章 結果…………………………………………………………….…...……24 一、田間及盆栽迷迭香病害的調查………………………………….……….25 二、迷迭香根腐病病原菌株之分離、鑑定與培養………………….…….…27 三、供試土壤及介質之消毒與除菌………………………………….……….29 四、迷迭香健康苗之繁殖…………………………………………….…….…30 五、根腐病分離株對迷迭香之病原性檢定…………………………..………32 （一）在試管中以無性繁殖扦插苗進行之病原性檢定……….……….32 （二）以穴盤苗進行之病原性檢定…………………………….…….…35 （三）接種後植株根部的再分離之結果………………………………..35 六、迷迭香根腐病菌之藥劑防治試驗……………………………….……….37 七、迷迭香根腐病菌對三種蔬菜幼苗的接種試驗………………….……….39 第五章 討論……………………………………………………………….……...42 一、田間及盆栽迷迭香病害的調查…………………………………..…..…..42 二、迷迭香根腐病病原菌株之分離、鑑定與培養………………….….……42 三、供試土壤及介質之消毒與除菌………………………………….….……43 四、迷迭香健康苗之繁殖…………………………………………….….……43 五、根腐病分離株對迷迭香之病原性檢定………………………….….……44 六、迷迭香根腐病菌之藥劑防治試驗……………………………….……….44 七、迷迭香根腐病菌對三種蔬菜幼苗的接種試驗………………….……….45 參考文獻…………………………………………………………………….….……46 附錄 迷迭香根腐病菌分離株的鑑定…………………………………….……...50 表目錄 表1、迷迭香地上部病害罹病度(Disease index)分級對照表..................................17 表2、花蓮縣田間迷迭香以茄子誘釣法進行分離株的初步鑑定...........................24 表3、六十盆迷迭香中所分離到的菌株種類與數量...............................................28 表4、分離株之20分群情形…………………………………………………….…29 表5、迷迭香試管扦插苗栽培中常見之腐生性病菌及發生比例...........................31 表6、迷迭香試管扦插苗於6/20日以20群各103/ml游走子接種後所發生之根腐比率..............................................................................................................................34 表7、迷迭香健康扦插苗在騰空穴盤接種20群病菌後所得之罹病度比率..........37 表8、迷迭香根腐病之藥劑防治試驗........................................................................38 表9、迷迭香根腐病之藥害試驗結果........................................................................39 表10、三種蔬菜幼苗接種迷迭香根腐病菌後6天後觀察根系褐化之結果...... ...39 表11、附錄分離所得菌株之鑑定結果.......................................................................52 圖目錄 圖1、花蓮縣吉安鄉某香草原迷迭香植株實地病害發生情形...............................16 圖2、迷迭香根腐病病根之病健部在解剖顯微鏡下的觀察情形...........................19 圖3、以5％ CV-8 + Ampicillin + Mycostatin + PCNB 選擇性培養基進行迷迭香根病健部組織分離情形..............................................................................................19 圖4、迷迭香穴盤苗的栽種情形...............................................................................22 圖5、由花市洽購迷迭香60盆在2007年5至6月之生長及發病情況...............25 圖6、迷迭香根腐病罹病度(Disease index)各級發病實物比較，由右至左為0及1至5級..........................................................................................................................26 圖7、由花市洽購迷迭香60盆在2007年5至6月之發病進展情況....................26 圖8、以5％ CV-8 + Ampicillin + Mycostatin + PCNB 選擇性培養基進行迷迭香根病健部組織分離培養1至兩天後的情形...............................................................28 圖9、試管中已發根迷迭香之扦插苗........................................................................31 圖10、迷迭香健康扦插苗以病原菌接種2天後根系發病情況..............................32 圖11、迷迭香健康扦插苗以病原菌接種4天後根系發病情況..............................33 圖12、迷迭香健康扦插苗以疫病菌接種2天後根部以甲基籃染色觀察之結果...33 圖13、迷迭香健康扦插苗在騰空穴盤接種病菌後罹病度之分級，由右至左為1至5級...........................................................................................................................36 圖14、迷迭香健康扦插苗在騰空穴盤接種病菌之對照組，皆無發病...................36 圖15、甘籃菜幼苗接種迷迭香根腐病菌6天後之結果...........................................40 圖16、番茄幼苗接種迷迭香根腐病菌6天後之結果..............................................40 圖17、包心芥菜幼苗接種迷迭香根腐病菌6天後之結果......................................41 圖18、 Ph-10(Phytophthora nicotianae)之形態........................................................60 圖19、 Ph-11(Phytophthora sp.)之形態....................................................................61 圖20、 Ph-10(Phytophthora citrophthora)之形態.....................................................62 圖21、. Py-2(Pythium sp.)之形態................................................................................63 圖22、 Py-11(Pythium sp.)之菌落形態.....................................................................64 圖23、 Py-12(Pythium group “P”)之形態.................................................................65 圖24、 Py-13(Pythium paligenes)之形態..................................................................66 圖25、 Py-16(Pythium sp.)之形態............................................................................67 圖26、 Py-32(Pythium sp.)之菌落形態....................................................................68 圖27、 Py-33(Pythium paligenes)之形態..................................................................69 圖28、 Py-37(Pythium oedochilum)之形態...............................................................70 圖29、 Py-45(Pythium myriotylum)之形態...............................................................71 圖30、 Py-89(Pythium sp.)之形態..............................................................................72 圖31、 Py-94(Pythium sp.)之形態.............................................................................73 圖32、 Py-104(Pythium sp.)之菌落形態...................................................................74 圖33、 Py-112(Pythium sp.)之菌落形態...................................................................75 圖34、 Py-117(Pythium sp.)之形態...........................................................................76 圖35、 Py-122(Pythium sp.)之形態...........................................................................77 圖36、 Py-130(Pythium catenulatum)之形態...........................................................78 圖37、 Py-132(Pythium catenulatum)之形態...........................................................792363935 bytesapplication/pdfen-US迷迭香根腐病腐霉菌疫病菌RosemaryRoot rotPythiumPhytophthoraotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/58082/1/ntu-96-R94633009-1.pdf