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標題: 菊花扦插苗細菌性軟腐病之研究與應用PCR技術偵測Erwinia chrysanthemi
Study on the bacterial soft rot of chrysanthemum cuttings and application of PCR technique for detection of Erwinia chrysanthemi
作者: 劉興隆
Liu, Hsing-Lung
關鍵字: chrysanthemum
bacterial soft rot
出版社: 植物病理學系
摘要: 自彰化縣田尾鄉菊花扞插苗細菌軟腐病罹病組織共分離到 32 個 Erwinia 軟腐細菌菌株, 經生理生化測定結果, 其中有 23 個菌 株為 Erwinia carotovora subsp. carotovora (簡稱 Ecc ), 其 餘 9 個菌株為 Erwinia chrysanthemi (簡稱 Ech ), 進一步鑑定 Ech 菌株得悉 8 個菌株屬於 subdivision Ⅱ及 biovar 6;一個菌株 屬於 subdivision Ⅳ及 biovar 3。 溫 度可影響菊花扦插苗軟腐病之發 生,在 25~30 ℃下 Erwinia 軟腐細菌引起之扦 插苗軟腐長度明顯較 15~20 ℃時為長,而其腐爛倒伏情形亦較嚴重。 此外介質 含水量愈高其 軟腐愈嚴重。又菊花插穗癒合時間愈長其軟腐病之發生也愈輕微。 以選 擇性培養基( modified CVP )偵測母株田之灌溉水、土壤、頂芽 Erwinia 軟腐細菌之存在情形, 結果顯示 Erwinia 屬軟腐細菌於此些標 本中出現之頻率 分別為 100%、15%、7%; 測試 41 個菊花栽培品種對 Erwinia 軟腐細菌之感受 性,結果顯示各品種間有差異,其中龍鳳小紅 最抗病,而芬蘭小粉最感病。藥劑 試驗結果顯示,菊花插穗基部以多保 鏈黴素及鏈四環黴素處理效果最好,可明顯 降低 Erwinia 軟腐細菌引 起之軟腐長度。 污染軟腐細菌之穴盤以鹽酸 (37%) 500 倍液或次氯酸 鈉 (Chlorox, 5%) 100 倍液處理, 於穴盤上即偵測不到軟腐 細菌。 此 外穴盤經 50 ℃溫水處理 1 小時,亦可完全殺死附在穴盤上面之軟腐 細 菌。此外應用蒸氣處理可有效殺死泥炭土中的軟腐細菌。以 5A/ 5B 引子 對應 用 PCR 偵測 Ech,結果顯示在 20 ul PCR 反應混合液中,可偵測 到之最低 Ech 在 150 ∼ 310 cfu 間。 又測試之部份非目標細菌於高濃 度時可影響以 5A/ 5B 引子對應用 PCR 偵測 Ech 之效率。利用 PCR 技 術可準確快速地偵測與鑑定 E. chrysanthemi。
A total of 32 strains of soft rot Erwinia were isolated from rotted tissues of chrysanthemum cuttings in Tienwei, Changhua. Based on the results of physiological and biochemical tests, 23 strains were identified as Erwinia carotovora subsp. carotovora (Ecc), while the other 9 strains were Erwinia chrysanthemi (Ech). According to the Dickey''s and Boccara''s systems, eight strains of Ech were classified to the subdibision II and biovar 6, while the other one strain was belong to the subdibision Ⅳ and biovar 3. Temperature could affect the soft rot severity of chrysanthemum cutting caused by soft rot Erwinia. Length of pith maceration of cuttings was significantly longer at 25~30 ℃ than that at 15~20 ℃. Water content of growing medium would also affect the soft rot development. Soft rot of chrysanthemum cuttings was more severe when the cuttings were planted in growing medium with higher water content. Severity of soft rot of chrysanthemum cuttings would be reduced if the cuttings were kept at high moist conditions for curing before planting. A modified CVP selective medium was used to study the occurrence of soft- rot Erwinia in mother stock field of chrysanthemum. Soft-rot Erwinia could be detected from irrigation water, soil and chrysanthemum shoot with frequency at 100%, 15% and 7%, respectively. Susceptibility of cultivars of chrysanthemum to Ecc and Ech was examined. Among the 41 cultivars tested, cultivar "Fen Lan Shiao Fen" was the most susceptible, while "Lung Fenq Hsiao Hung" was the least susceptible. Among the agrochemicals tested, thiophanate methyl + streptomycin and streptomycin + tetracycline appeared to be the most effective to protect the chrysanthemum cutting from the infection of soft rot Erwinia. Cells of soft-rot Erwinia could not survive in trays treated with HCl (37%) 500x or Chlorox (5%) 100x for 2~3 min. They also could not survive in trays immersed in 50 ℃ hot water for 1 hr. Furthermore, cells of soft rot Erwinia in peat moss could be killed by the steam treatment. PCR technique was used to detect the Ech in soft rot tissues of plants. Sensitivity of PCR for detection of Ech with primer pair 5A/ 5B was approximately 150-310 cells in 20 ul PCR reaction mixture. Non- target bacteria tested did not affect the efficiency of amplification of Ech DNA in PCR assay. It appeared that cells of Ech in soft rot tissues in plants could be detected efficiently and rapidly by the PCR with primer 5A/ 5B.
Appears in Collections:植物病理學系



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