臺灣博碩士論文加值系統

炭疽病 (Anthracnose) 為目前台灣草莓 (Fragaria × ananassa Duchessne) 生產過程中極具威脅之重要病害，常會造成全株之死亡。而若能檢測及監測田間病原菌之感染源及其密度，將有助於此一病害之預防及控管。因此，本研究目的之一即為草莓炭疽病菌半選擇性培養基之研發，希望利用此選擇性培養基，可順利從草莓植株、土壤及其他植物中檢測出炭疽病菌。經比較 25 種抗微生物藥物對炭疽病菌及其他常見雜菌之抑制效果，從中挑選可抑制雜菌而較不抑制炭疽病菌者，加以組合及實測，發現以免賴得 (Benomyl，5 mg/L)、白克列 (Boscalid，10 mg/L)、依普同 (Iprodione，5 mg/L)、四環黴素 (Tetracycline hydrochloride，50 mg/L) 及滅達樂 (Metalaxyl，10 mg/L)，加入馬鈴薯葡萄糖瓊脂培養基 (PDA) 中配製成之半選擇性培養基 (簡稱PDA-BBITM)，可有效自草莓植株及雜草葉片上分離到炭疽病菌，且相較於前人使用之培養基，可大幅減少雜菌之干擾。利用此培養基亦可自內外部皆無病徵之草莓苗冠部檢測出炭疽病菌，應用此培養基於田間生產之草莓苗炭疽病菌之篩檢，發現檢出之帶菌率與定植後之補植率有一定程度之關連，故未來 PDA-BBITM 應可應用於健康草莓苗之篩檢。而從土壤檢測炭疽病菌時，發現亦可使用相同之抗微生物藥劑組合，但改以果膠作為碳源的查氏培養基 (Czapek-Dox agar) 取代 PDA，較有利於減少雜菌之干擾。以此培養基檢測炭疽病菌孢子於土壤中之殘存，發現其存活率會於 60 天內下降至 30% 以下，日曬及淹水之處理則會加速其下降，而土壤中之罹病植物殘體則於 70 天時幾乎檢測不到炭疽病菌，因此推測炭疽病菌於氣候濕熱之台灣應無法於土壤中長時間殘存。將炭疽病菌接種於 11 種雜草上，發現接種後均未出現明顯之病徵，然而至接種後 28 天時，大部分植物仍可檢測到炭疽病菌。另檢測田間採集之雜草，發現僅光風輪 (Clinopodium gracile (Benth.) Kuntze) 及牛筋草 (Eleusine indica (L.) Gaertn.) 上可檢測到對草莓具病原性之炭疽病菌。最後，為比較現行藥劑及拮抗微生物對草莓炭疽病之防治效果，並測試可行之其他防治藥劑，本研究以盆栽草莓測試待克利 (Difenoconazole)、腐絕快得寧 (Thiabendazole + Oxine-copper)、百克敏 (Pyraclostrobin)、平克座 (Penconazole)、賽福座 (Triflumizole)、鏈黴菌 (Streptomyces sp.) Yu-01 及枯草桿菌 (Bacillus subtilis) 之防治率，結果顯示百克敏之防治效果最佳。

Anthracnose (Colletotrichum) is a severe disease of strawberry (Fragaria × ananassa Duchessne) in Taiwan, which often results in wilting of plants. The capability of detecting and monitoring the inoculum sources and density in the field would aid in prevention and control of this disease. Thus, this research is aimed to develope a semi-selective medium for pathogens of strawberry anthracnose, and to apply it to detect Colletotrichum in strawberry plants, soil and other plants. By comparing the inhibition effects of 25 antimicrobial chemicals on Colletotrichum and major contaminating microbes, and selecting those inhibiting the latter but not the former, it was found that PDA-BBITM (potato dextrose agar (PDA) with 5 mg/L benomyl, 10 mg/L boscalid, 5 mg/L iprodione, 50 mg/L tetracycline hydrochloride, and 10 mg/L metalaxyl) could effectively isolate Colletotrichum from strawberry plants and weeds with less disturbance from the contaminating microbes compared with previously used semi-selective media. Besides, this medium could also detect Colletotrichum from crowns of symptomless strawberry seedlings. Applying this medium to detect Colletotrichum in field-grown seedlings, it showed that the detection rate is positively associated with the replanting rate. Thus, it is suggested that PDA-BBITM can be applied in the system of healthy seedlings certification. For detecting Colletotrichum in soil, the same combination of antimicrobial chemicals could also be used, but the PDA medium needed to be replaced with Czapek-Dox agar, modified by using pectin as the carbon source, to reduce the disturbance of contaminating microbes. By this modified Czapek-Dox agar, it was discovered that the survival rate of Colletotrichum conidia in soil would be less than 30% after 60 days, while solarization and flooding could reduce the survival rate rapidly. In addition, diseased debris could hardly be detected with Colletotrichum after buried in soil for 70 days, indicating that Colletotrichum might not be likely to survive in soil for a long time in wet and hot climate in Taiwan. None of 11 tested weeds developed anthracnose symptoms after inoculated with Colletotrichum conidia, but most of them could be detected with Colletotrichum at 28 days after inoculation. We also found that the weeds collected from strawberry growing fields, including Clinopodium gracile (Benth.) Kuntze and Eleusine indica (L.) Gaertn., could be detected with Colletotrichum pathogenic to strawberries. Finally, to compare and evaluate the control effeciency of fungicides and antagonistic microoragnisms to strawberry anthracnose, difenoconazole, thiabendazole + oxine-copper, pyraclostrobin, penconazole, triflumizole, Streptomyces sp. Yu-01 and Bacillus subtilis were tested in pot tests, and the results showed that pyraclostrobin exhibited the highest control rate among them.

口試委員會審定書............................................................................................................i
誌謝...................................................................................................................................ii
中文摘要..........................................................................................................................iii
Summary............................................................................................................................v
目錄..................................................................................................................................vii
表目錄..............................................................................................................................xii
圖目錄..............................................................................................................................xiv
第一章 前言.................................................................................................................1
一、草莓栽培概說.........................................................................................................1
二、草莓之基本特性及栽培管理.................................................................................1
三、台灣地區草莓之相關病害.....................................................................................2
四、研究目的.................................................................................................................3
第二章 前人研究............................................................................................................4
一、感染草莓之炭疽病菌種類....................................................................................4
二、草莓炭疽病之病徵及危害....................................................................................5
三、草莓炭疽病菌於土壤中之殘存............................................................................6
四、草莓炭疽病菌於雜草寄主與其他作物上之存活................................................6
五、草莓炭疽病菌之檢測技術....................................................................................7
六、炭疽病菌選擇性培養基之研究.............................................................................7
七、草莓炭疽病防治之研究.........................................................................................8
第三章 材料與方法.........................................................................................................11
一、草莓炭疽病相關菌株之分離及鑑定.................................................................11
(一) 草莓炭疽病菌菌株之分離及保存...........................................................11
(二) 萎凋草莓冠部雜菌菌株之分離及鑑定...................................................12
二、半選擇性培養基之藥劑篩選..............................................................................14
三、利用半選擇性培養基檢測草莓植株上炭疽病菌之效果..................................16
(一) 四種半選擇性培養基檢測草莓病株冠部炭疽病菌之效果...................16
(二) 利用 PDA-BBITM 半選擇性培養基檢測田間草莓炭疽病株.............17
四、半選擇性培養基檢測土壤中草莓炭疽病之效果..............................................17
(一) 利用不同營養源之 7 種 ASBBIE 培養基檢測土壤中草莓炭疽病之效果....................................................................................................................17
(二) 比較 pectin-ASBBIE 及 BBITM二種半選擇性培養基檢測土壤中草莓炭疽病之效果................................................................................................18
(三) 應用 pectin-BBITM 檢測田間土壤中之炭疽病菌...............................18
五、BBITM 半選擇性培養基對炭疽病菌影響之測試...........................................19
(一) BBITM 半選擇性培養基對草莓炭疽病菌分生孢子發芽抑制之測試..19
(二) 33 株草莓炭疽病菌菌株於 BBITM 半選擇性培養基上生長情形之比較......................................................................................................................19
(三) 其他作物分離之5 種炭疽病菌菌株於 BBITM 半選擇性培養基上之生長情形............................................................................................................19
六、應用 PDA-BBITM 半選擇性培養基檢測帶病草莓苗之研究........................20
(一) 草莓苗潛伏感染炭疽病菌之檢測...........................................................20
(二) 應用 PDA-BBITM 半選擇性培養基檢測田間草莓苗炭疽病潛伏感染之情形................................................................................................................20
七、應用pectin-BBITM半選擇性培養基研究炭疽病菌於土壤中之殘存情形....21
(一) 不同土壤及含水率對炭疽病菌孢子殘存之影響...................................21
(二) 太陽能加熱及淹水對炭疽病菌之孢子於土壤中殘存之影響...............22
(三) 草莓炭疽病菌於土壤中罹病殘體之殘存測試.......................................22
八、草莓炭疽病菌田間其他寄主之調查與研究......................................................23
(一) 應用 PDA-BBITM檢測炭疽病菌人工接種到雜草上之存活情形.......23
(二) 應用 PDA-BBITM檢測草莓田雜草上之炭疽病菌...............................23
九、炭疽病菌分離株對草莓之病原性檢測..............................................................24
十、草莓炭疽病防治之研究......................................................................................25
(一) 生物防治資材抑制草莓炭疽病效果之研究...........................................25
(二) 化學藥劑抑制草莓炭疽病效果之研究...................................................26
(三) 化學藥劑及生物防治資材防治草莓炭疽病效果之研究.......................26
第四章 結果.....................................................................................................................30
一、草莓炭疽病相關菌株之分離及鑑定..................................................................30
二、半選擇性培養基之藥劑篩選..............................................................................33
三、利用半選擇性培養基檢測草莓植株上炭疽病菌之效果..................................41
(一) 四種半選擇性培養基檢測草莓病株冠部炭疽病菌之效果...................41
(二) 利用 PDA-BBITM 半選擇性培養基檢測田間草莓炭疽病株.............44
四、半選擇性培養基檢測土壤中草莓炭疽病之效果..............................................48
(一) 利用不同營養源之7種ASBBIE培養基檢測土壤中草莓炭疽病之效果........................................................................................................................48
(二) 比較 pectin-ASBBIE及BBITM二種半選擇性培養基檢測土壤中草莓炭疽病之效果....................................................................................................57
(三) 應用 pectin-BBITM 檢測田間土壤中之炭疽病菌...............................57
五、BBITM 半選擇性培養基對炭疽病菌影響之測試...........................................59
(一) BBITM 半選擇性培養基對草莓炭疽病菌分生孢子發芽抑制之測試..59
(二) 33 株草莓炭疽病菌菌株於 BBITM 半選擇性培養基上生長情形之比較......................................................................................................................62
(三) 其他作物分離之5 種炭疽病菌菌株於 BBITM 半選擇性培養基上之生長情形............................................................................................................66
六、應用PDA-BBITM半選擇性培養基檢測帶病草莓苗之研究...........................67
(一) 草莓苗潛伏感染炭疽病菌之檢測...........................................................67
(二) 應用 PDA-BBITM 半選擇性培養基檢測田間草莓苗炭疽病潛伏感染之情形................................................................................................................69
七、應用pectin-BBITM半選擇性培養基研究炭疽病菌於土壤中之殘存情形.....71
(一) 不同土壤及含水率對炭疽病菌孢子殘存之影響...................................71
(二) 太陽能加熱及淹水對炭疽病菌之孢子於土壤中殘存之影響...............72
(三) 草莓炭疽病菌於土壤中罹病殘體之殘存測試.......................................74
八、草莓炭疽病菌田間其他寄主之調查與研究......................................................75
(一) 應用 PDA-BBITM檢測炭疽病菌人工接種到雜草上之存活情形.......75
(二) 應用 PDA-BBITM檢測草莓田雜草上之炭疽病菌...............................76
九、炭疽病菌分離株對草莓之病原性檢測..............................................................82
十、草莓炭疽病防治之研究......................................................................................86
(一) 生物防治資材抑制草莓炭疽病效果之研究...........................................86
(二) 化學藥劑抑制草莓炭疽病效果之研究...................................................87
(三) 化學藥劑及生物防治資材防治草莓炭疽病效果之研究.......................88
第五章 討論.....................................................................................................................93
一、草莓炭疽病相關菌株之分離及鑑定..................................................................93
二、半選擇性培養基檢測草莓植株上炭疽病菌之效果..........................................93
三、BBITM 半選擇性培養基對不同地區及來源之炭疽病菌之適用性...............95
四、應用PDA-BBITM半選擇性培養基檢測帶病草莓苗之研究...........................99
五、半選擇性培養基檢測土壤中草莓炭疽病菌之效果........................................101
六、應用pectin-BBITM半選擇性培養基研究炭疽病菌於土壤中之殘存情形...102
七、草莓炭疽病菌田間其他寄主之調查與研究....................................................104
八、草莓炭疽病防治之研究....................................................................................106
參考資料........................................................................................................................108
附錄一............................................................................................................................116

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