向日葵細菌性軸腐病知特性、品種抗性及藥劑篩選

[[abstract]]民國91 年6 月，在苗栗縣大湖?區所種植之向日葵發生植株葉片乾枯萎凋的徵狀，但莖仍直立於田間，切開莖部，其內部已成?空且具水浸狀腐爛，若發生於植株莖苗期被害，嚴重者倒伏死亡。92 年夏?同樣情形再度發生。經柯霍氏法則確認該病原為細菌。經生理生化，Biolog 鑑定及專?性引子進行PCR 分析等測定，確認該病原菌包括Erwinia chrysanthemi (Ech) 及E. carotovora pv.carotovora (Ecc)。該病原菌主要造成向日葵植株軸心?空及腐爛，進而引起植株萎凋枯死，因此稱為向日葵細菌性軸腐病（stalk rot）。由Ecc 及Ech 引起之向日 葵細菌性軸腐病為台灣向日葵首次記錄。品種抗性測定發現供試九種向日葵品種?以東北八重最為感病，月光及光輝雖感病但抗性較強。軸腐病菌對馬鈴薯塊莖、大白菜梗、胡蘿蔔塊根均具強致腐能力。測試市售11 種藥劑在?般使用濃度?以濾紙圓盤法測試對軸腐病菌菌株生長之抑制效果，顯示除?基鋅乃浦對該病菌之生長無抑制作用外，其餘 10 種包括鏈黴素、?環黴素、鏈?環黴素、嘉賜黴素、多保鏈黴素、氫氧化銅、鹼性氯氧化銅、嘉賜銅、鋅錳乃浦及鋅錳滅達樂等藥劑均能抑制其生長，且以?環黴素藥劑之效果最佳

細菌性葉斑病菌Pseudomonas cichorii專一性引子之開發

[[abstract]]以60個逢機引子應用隨機增幅多型性核酸技術(RAPD)增幅並篩選出對細菌性葉斑病菌Pseudomonas cichorii (Swingle) Stapp特有之1,100 bp的專一性片段，進一步將該片段接入Topo載體pCR(上? @)Ⅱ-TOPO得到選殖株，並分析其核酸序列，再根據其序列設計出對Pseudomonas cichorii具專一性之引子對SfL1/SfR2。應用此引子對進行聚合酵素連鎖反應(polymerase chain reaction，PCR)，其對供試向日葵細菌性葉斑病菌皆能增幅出379 bP的片段，但對供試之6屬21種其他非標的之病原菌及雜菌菌株則不會產生任何片段。當以引子對SfL1/SfR2測試細菌性葉斑病菌P. cichorii之全DNA時，其靈敏度可偵測到5~10pg，而用於偵測其細菌數之靈敏度則可以測到5.5~9個活菌數。將向日葵細菌性葉斑病菌(P. cichorii)10^6cfu/ml菌液與其他細菌菌株之菌液10^5~10^8cfu/ml混合，再以引子對SfL1/SfR2進行聚合酵素連鎖反應，結果顯示不影響其對P. cichorii之偵測效率。應用單一菌落快速檢定法可於3~4hr內鑑定向日葵細菌性葉斑病菌，該引子對可用於人工接種葉斑病菌之向日葵植株樣品之偵測，因此確認所設計之引子對SfL1/SfR2可用於快速鑑定及診斷細菌性葉斑病菌P. cichorii。 A specific PCR (polymerase chain reaction) primer pair have been developed using RAPD (random amplified polymorphic DNA) to detect Pseudomonas cichorii. Totally sixty random primers were used to find specific DNA fragments of P. cichorii, and a specific DNA fragment of 1,100 bp amplified by the primer OPX17 was cloned into the pCR(superscript @)Ⅱ-TOPO vector and further sequenced to design a specific primer pair SfL1/SfR2. The primer pair could amplify a distinct band of 379 bp that was specific to P. cichorii, and no DNA fragment amplified by the same primer pair from the other tested 21 bacterial species in 6 genera. Sensitivity of PCR for detection of P. cichorii with primer pair SfL1/SfR2 was between 5~10 pg for purified DNA and 5.5~9 cfu for cultured cells. Non-target bacteria did not affect the efficiency of specific amplification of P. cichorii in PCR assay with primer pair SfL1/SfR2. PCR technique using primer pair SfL1/SfR2 identifies the culture of P. cichorii within 3-4 hours and detected the bacterium in artificially inoculated sunflower leaf tissue. Based on the data provided, we conclude that the primer pair SfL1/SfR2 might be a useful tool for rapid identification and diagnosis of P. cichorii

台灣火鶴花細菌性葉枯病菌(Xanthomonas axonopodis pv. Dieffenbachiae)之血清偵測

[[abstract]]以菌體固定法將葉枯病菌（Xanthomonas axonopodis pv. dieffenbachiae）A008及A072二個菌株 製備成抗原，分別注射於紐西蘭大白兔內製作多元抗體，再以此二種抗血清測試臺灣火鶴花葉枯病 菌，結果顯示臺灣火鶴花葉枯病菌具有至少二種之血清型（serovar）。以瓊脂免疫擴散法測試，發 現A008抗血清只與由台灣分離之葉枯病菌177個供試菌株中之A003及A008二個菌株形成明顯反應帶， 所佔比率為 1.1 %，而A072抗血清則與供試菌株中之168個菌株間均形成明顯反應帶，所佔比率為 94.9 %。以瓊脂免疫擴散法測試，此二種抗血清與供試之Agrobacterium tumefaciens、Erwinia carotovora subsp. carotovora、E. chrysanthemi、E. herbicola、E. herbicola pv. gypsophilae 、Pseudomonas fluorescens、P. putida、Ralstonia solanacearum、Xanthomonas albilineans、X. campestris. pv. campestris、X. c. pv. citri、X. c. pv. glycines、X. c. pv. mangiferaeindicae、X. c. pv. phaseoli、X. c. pv. uppalii、X. c. pv. vesicatoria、X. fragariae及X. oryzae pv. oryzae等菌株均無反應，但以直接酵素聯結抗體免疫法 （direct DAS-ELISA）測試時，則顯示A008抗血清與來自柑橘之 X. c. pv. citri 菌株有反應， 而A072抗血清則與來自彩色海芋之 E. c. subsp. carotovora 菌株有反應。利用direct DAS-ELISA 可檢測到104 cfu/ml菌量之抗原。將接種A072之火鶴花病葉直接以direct DAS-ELISA方法進行偵測， 則可檢測的菌量約106 cfu/ml。若以ET培養基（esulin-trehalose medium）先增量培養後再利用 direct DAS-ELISA方法進行偵測，則可檢測的菌量約102 cfu/ml

鑑別Erwinia carotovra subsp. carotovira及Erwinia chrysanthemi細菌性軟腐病菌專一性引子之開發

[[abstract]]利用隨機增幅多型性核酸技術(RAPD)增幅彩色海芋細菌性軟腐病菌菌株Erwinia carotovora subsp. Carotovora (Ecc)之基因體DNA，篩選出一1,200 bp具專一性之片段Y20，該片段經選殖後進行轉型質體pY20Ecc-1200bP核酸序列分析，再依據其序列與NCBI核酸資料庫比對得到該序列與E. chrysanthemi (Ech)之rhi N基因有83%相似處，並根據相同序列處設計出專一性引子對Ec3F/Ec4R，可用於區分Ecc及Ech。應用此專一性引子對進行聚合酵素連鎖反應，對於供試225株Ecc菌株均可增幅出497bP之片段，供試85株Ech菌株均可增幅出548bp之片段，但對供試之其他非標的菌株皆無法增幅出任何片段。以該引子對偵測彩色海芋細菌性軟腐病菌Ecc及蝴蝶蘭軟腐病菌Ech之DNA時，靈敏度同樣為10~50 pg，且與6屬7種其他非標的菌株DNA混合時並不影響其偵測結果。以Ec3F/Ec4R偵測Ecc及Ech菌液之靈敏度時，最低分別可測到1.2×10^1及1.1×10^1個細菌數，且與其他2屬3種其他非標的細菌之菌液混合時並不會影響其偵測結果。以Ec3F/Ec4R建立對於EcC及Ech之單一菌落快速檢定法，確實可偵測並區分Ecc及Ech菌株，且可應用於田間軟腐病株之偵測，因此，確認本試驗所設計之引子對Ec3F/Ec4R能快速鑑定及偵測軟腐病菌，並能區分Ecc及Ech。 A specific PCR (polymerase chain reaction) primer pair has been developed using RAPD (random amplified polymorphic DNA) to differentially detect Erwinia carotovora subsp. carotovora (Ecc) and E. chrysanthemi (Ech). A specific 1,200 bp DNA fragment was amplified and cloned from Ecc DNA using OPY2O primer, one of the sixty random primers used in RAPD. Nucleotide sequence of this 1,200 bp DNA showed 83% similarity to Ech rhiN gene, where conserved sequences were displayed in two regions. But in Ech rhiN sequence, there are additional 49 bp nucleotides inserted between these two conserved regions. Herein, a set of primers Ec3F/Ec4R was designed and applied to differentially detect Ecc and Ech by PCR. Using bacterial chromosomal DNA as templates, a distinct 497 bp fragment can be amplified from 225 Ecc strains, and a 548 bp fragment was amplified from 85 Ech strains. No fragment can be amplified from the other seven bacterial species in six genera. Sensitivity of PCR for detecting Ecc and Ech was between 10~50 pg for purified DNA and 1.1×10^1 cfu and 1.2×10^1 cfu for cultured cells, respectively. It took 3-4 hr to detect Ecc and Ech by colony PCR using Ec3F/Ec4R primer set and the target bacteria in infected tissues of cala lily and phalaenopsis. Results presented here suggest that the primer pair Ec3F/Ec4R applied in PCR can be very useful in rapid identification, detection, and differentiation of the soft rot pathogens Ecc and Ech

橫山梨果腐病之病因及病原菌藥劑感受性測定

[[abstract]]於1998 年6 月間在南投縣中寮鄉種植之橫山梨發現果實腐爛問題，經柯霍氏法則確認該病原為細菌，隔年（1999）於新竹栽培之橫山梨亦發現相同病害。此病害主要危害果實，在枝條及葉片上不引起病徵，果實初期病徵僅在表皮上出現淡褐色水浸狀小斑點，病斑繼續擴展造成內部果肉腐爛，輕壓表皮具彈性，嚴重時整個果實腐爛，並有乳白色汁液與氣泡湧出。從罹病果實上分離之細菌，經生理生化測定，Biolog 鑑定系統，專一性引子對PCR 分析等方法，鑑定該病原細菌為Erwinia carotovora subsp. carotovora。將此病定名為果腐病。以蟲針沾細菌懸浮液刺傷果實及浸菌無傷口接種等方法接種橫山梨果實，發現僅傷口處理者出現病徵，且病徵擴展迅速，病斑直徑可達1.2 cm/day，該病菌也感染新興梨，在馬鈴薯、胡蘿蔔及洋蔥等組織上具有致腐能力，且可造成煙草植株腐爛。果實蠅媒介試驗顯示在有果實蠅成蟲之蟲箱內，不論以病菌處理果實蠅取食之洋菜膠或以病菌直接覆於果實上，供試之橫山梨及新興梨果實均出現典型果腐病徵。另外，讓果實蠅於梨果實上產卵後，於產卵孔滴病原菌也產生相同果腐病徵。測試市售11 種藥劑在一般使用濃度下對該病菌生長之抑制效果，結果顯示除鋅乃浦外，其餘供試之枯萎寧、四環黴素、鏈黴素、安達菌、嘉賜黴素、嘉賜銅、氫氧化銅、鹼性氯氧化銅、銅合浦及鋅錳乃浦等藥劑於培養基上均能抑制該病菌之生長

Burkholderia andropogonis引起之檳榔葉斑病及藥劑篩選。

[[abstract]]民國95年間於台中縣大坑、嘉義縣阿里山鄉、屏東縣高樹鄉、高雄縣美濃鎮及花蓮縣鳳林鎮等地種植之檳榔藥片上發生褐化壞疽病斑，病斑周圍伴有黃暈，病斑隨著葉脈擴散呈不規則形，由柯霍氏法則、生理生化、Biolog鑑定及脂肪酸組成分析結果顯不供試檳榔病原菌株?Burkholderia andropogonis，進一步應用對B. andropogonis具有專一性之引子對進行PCR反應，結果由該病原菌可增幅出410bp之專一性DNA條帶，並且以16S rDNA進行定序與鑑定，確認該病原菌?B. andropogonis，由其引起之病徵定名?檳榔葉斑病(bacterial leaf spot of betel palm)。以市售12種藥劑測試在一般使用濃度下對該病菌生長之抑制效果，顯示12種藥劑均能抑制病原菌於PDA培養基上的生長，其中又以四環黴素效果最佳，依其抑制效果依序?四環黴素、歐索林酸、嘉賜黴素、嘉賜銅、氫氧化銅、鏈四環黴素、多保鏈黴素、三元硫酸銅、鋅錳乃浦、鏈黴素、鹼性氯氧化銅及鋅錳滅達樂等。 In 2006, a leaf spot disease was found on betel polio in several locations in Taiwan. The infected leaves showed irregular brown necrotic spots surrounded by yellowish halo. A grain negative, rod shaped bacterium was consistently isolated front the diseased tissues. The bacterium was identified as Burkholderia andropogonis based on its physiological characteristics, the Biolog GN MicroPlate system, the MIS system for cellular fatty acid analysis. polymerase chain reaction (PCR) with a pair of primers specific for B. andropogonis, 16S rDNA sequence and pathogenicity tests. In vitro screening for the efficacy of different agrochemicals to inhibit bacterial growth on PDA plates showed that all tested chemicals, including copper bactericides, antibiotics, oxolinic acid, and carbamates. were effective. Among them, tetracycline was most effective

榕樹細菌性癌腫病菌Agrobacterium tumefaciens在台灣之發生

[[abstract]]於田尾地區田間及盆栽栽培之金門榕(Ficus microcarpa L. f. var. pusilifolia Liao cv. Kin-men)，中葉榕(Ficus microcarpa L. f. cv. Middle-leaf)及屏東地區之小葉榕(Ficus microcarpa L. f. var. pusilifolia Liao)植株上發現腫瘤病，腫瘤大小可達10-20餘公分，主要發生於根冠、樹幹或枝條之修剪處或其它傷口處。由腫瘤處分離所得之細菌在PDA培養基上形成黏濕，圓形具亮光，不透明的白色菌落，為革蘭氏陰性，好氣性，具有1-6根週生鞭毛的桿狀細菌。該細菌以Biolog GN Microplate鑑定結果與Agrobacterium tumefaciens相近，再依選擇性培養基及各種生理生化試驗結果屬於biovar 1。該細菌接種於胡蘿蔔肉質根切塊上，經4-5天後於形成層處形成淡綠色癒合組織狀之腫大，但未出現根狀增生；以針刺及剪枝條的方法接種於盆栽金門榕及中葉榕之枝條上及農友301番茄植株之莖或葉柄處，榕樹於接種後約三星期於接種處出現淡黃色小腫瘤，顏色隨著腫瘤增大而加深，而番茄植株則於接種後7-10天於接種處形成淡白綠色之腫瘤，之後瘤漸增大且顏色轉為淡黃色，整個瘤約可大到1公分，且在近感染處同側的葉片呈現失水狀，該葉片最後萎凋。根據上述試驗結果顯示造成榕樹癌腫病之病原菌為Agrobacterium tumefaciens biovar 1