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標題: 拮抗性鏈黴菌S2菌株在十字花科細菌性斑點病防治應用之發展研究
Development of Antagonistic Streptomyces sp. S2 for Controlling Bacterial Leaf Spot Disease of Crucifers Caused by Xanthomonas campestris pv. raphani
作者: 吳昱陞
Yu-Sheng Wu
關鍵字: 鏈黴菌;十字花科;細菌性斑點病;細菌性葉斑病;液態培養;生物防治;Streptomyces;crucifers;bacterial leaf spot disease;submerge culture;biological control
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十字花科細菌性斑點病由Xanthomonas campestris pv. raphani (Xcr)所引起,近年來已陸續在許多國家報導於十字花科、茄科以及葫蘆科等作物上發現。本次研究將由罹病甘藍上所分離到的分離株,經過柯霍氏法則回接、16S-23S rRNA區間序列分析及病原型檢測,證實病原菌為X. campestris pv. campestris (Xcc)與Xcr。為了選出有潛力的拮抗性鏈黴菌,以Xcr和多種細菌與真菌病原為拮抗標靶,經由拮抗對峙篩選後,結果顯示鏈黴菌S2的拮抗圖譜最廣泛,在對峙試驗中可抑制細菌、卵菌、子囊菌與擔子菌等所有供試病原菌。生物防治菌必須對植物與人類具安全性,因此將鏈黴菌S2利用16S rRNA基因序列分析,S2與S. andamanensis strain KC-112的16S rDNA序列相似度達99.8%,同時與造成馬鈴薯瘡痂病的鏈黴菌類群(scab-causing strains)在親緣關係樹分析中可以明確的分在不同類群。為了更進一步證實S2菌株的生物安全性,使用專一性引子對TxtA1/TxtA2、Nf/Nr及Tom3/Tom4檢測,鏈黴菌S2不帶有致病相關基因txtA (thaxtomin synthetase A)、nec1 (necrotic protein),以及tomA (tomatinase)。此外,利用串聯式液相層析質譜儀,以及甘藍和蘿蔔幼苗來檢測S2培養液,證實S2不產生thaxtomin A並且對植物無毒害影響。綜合分子生物技術檢測方法與發酵液代謝物分析的結果,證實鏈黴菌S2對於植物沒有毒性,符合生物防治菌的安全性。為了能有效進行生物防治試驗,必須先調整適合S2的液態培養配方。在鏈黴菌培養常用的ISP培養基之中,S2在ISP2固態培養基上生長速度最佳且產孢良好。接著在S2的碳氮源利用性分析中,S2對發酵常用原料葡萄糖、木糖、麥芽糖及乳糖的利用性佳,對蔗糖的利用性較差,而對澱粉幾乎不利用。S2除了對硝酸鈉的利用性較差之外,對其餘測試的氮源皆生長良好。綜合ISP與碳氮源分析的結果,將ISP2配方的葡萄糖以其他糖類置換後,測試對於生長的影響。S2於添加麥芽糖的ISP2培養基(mISP2)能夠在液態培養下產孢。以mISP2為基礎進行一系列配方調整測試,如麥芽糖的濃度、酵母抽出物與麥芽抽出物的比例、鈣離子含量、磷酸緩衝液的濃度以及初始pH值等條件,目前結果仍以mIPS2為最佳的配方。當鈣離子濃度高於100 ppm或磷酸鹽濃度高於0.005 M,對S2的液態產孢都有不良影響。S2在mISP2液態培養液中產生的孢子,也經由穿透式電子顯微鏡觀察孢子構造,證實與在固態培養的成熟孢子一樣具有加厚的細胞壁。在細菌性斑點病的防治試驗中,使用S2培養於mISP2養液5天的培養液,以10倍稀釋的S2培養液浸泡能有效降低帶菌種子(105 CFU/ml Xcr PC9)的發病率;噴施100倍稀釋的S2培養液能有效降低接種106 CFU/ml Xcr PC9的葉片罹病度。在種子與葉面防治試驗中,S2能夠與歐索林酸混合使用而沒有不良的影響。本研究顯示,鏈黴菌S2具安全性與拮抗廣譜性。在mISP2液態中培養能夠正常生長與產孢,並能夠在溫室試驗中有效的防治甘藍細菌性斑點病,S2還對多種藥劑低敏感性,極具有潛力開發成為生物製劑,未來能應用於綜合病害管理體系,藉此降低化學藥劑的使用與輔助抗藥性管理等,減少對環境的衝擊。

Bacterial leaf spot disease of crucifers is caused by Xanthomonas campestris pv. raphani (Xcr). In recent years, it has been reported on crucifers, solanaceous crops, and cucurbits in many countries. In this study, we had found and collected diseased cabbage seedlings from the nursery. According to the results of re-inoculation, 16S-23S rRNA spacer sequence analysis, and detection of Xanthomonas campestris pathovars, the isolates from diseased seedlings were identified as Xanthomonas campestris pv. campestris (Xcc) and Xcr. To develop potential biocontrol agents, screening antagonistic Streptomyces species against with Xcr, other bacterial and fungal pathogens had done by dual culture. Streptomyces sp. S2 was selected as a broad-spectrum antagonist against with tested bacteria, oomycetes, ascomycetes and basidiomycetes. It's important that potential biocontrol agents have to be harmless for plants and humans. Therefore, Streptomyces sp. S2 was identified by 16S rRNA gene sequence analysis and S2 showed 99.8% 16S rDNA sequence identity to that of S. andamanensis strain KC-112 which appeared to be phylogenetically distinct from scab-causing strains. Furthermore, pathogenicity factors such as thaxtomin synthetase A (txtA), necrotic protein (nec1), and tomatinase (tomA) had not detected in S2 genome by using specific primers TxtA1/TxtA2, Nf/Nr, and Tom3/Tom4 respectively. In addition, S2 cultured broth was analyzed for thaxtomin A by using LC/MS/MS and seedling assay. As a result, S2 didn't produce thaxtomin A, and no phytotoxicity symptom was observed on the cabbage and radish seedlings treated with S2 cultured broth. In brief, Streptomyces sp. S2 as a potential biocontrol agent is safety for application. To control bacterial leaf spot disease effectively, culture condition of S2 had established. The characteristic of S2 grown on ISP medium were analyzed, in consequence, the growth and sporulation of S2 was best on ISP2 medium. Following utilization of carbon and nitrogen source analysis, S2 has good utilization of glucose, xylose, maltose and lactose, poor utilization of sucrose, and no utilization of starch among materials commonly used to fermentation. All of nitrogen sources tested could be utilized well by S2, but sodium nitrate. Furthermore, the effect on replacement of glucose in ISP2 medium with different carbohydrates was evaluated. The sporulation of S2 in submerged ISP2 medium modified by replacing glucose with maltose (mISP2) could be observed under microscopy. Based on mISP2 medium, different concentration of maltose in mISP2, different ratio of yeast extract to malt extract, different concentration of calcium and phosphate buffer, and initial pH value were evaluated. The result so far, mISP2 medium has been the best recipe for sporulation of S2 in submerged culture with initial pH 6.0-7.0; it was negative effect on growth and sporulation when the amount of calcium ion more than 100 ppm and phosphate buffer more than 0.005M in mISP2. To confirm the spores produced in submerged mISP2 were mature and intact, spores collected from submerged culture were compared with spores from agar plates by using electron transmission microscopy. The thick spore cell wall and nozzles could be observed in solid and submerged culture. Finally, 5-dsy mISP2 cultured broth of S2 was used to control disease caused by Xcr on cabbage seeds and leaf. The disease incidence of contaminated seeds(105 CFU/ml) was significantly reduced by treating with the 10×-diluted S2 cultured broth. As well as, the disease severity of cabbage leaves inoculated with Xcr(106 CFU/ml) was significantly reduced by spreading the 100×-diluted S2 cultured broth on leaf surface. In addition, S2 cultured broth could be mixed with bactericide oxolinic acid 100 ppm without negative effect on disease control. In conclusion, Streptomyces sp. S2 is a safe and broad-spectrum antagonist against phytopathogens. Streptomyces sp. S2 grew well and formed spores in mISP2 submerged culture. In greenhouse trial, bacterial leaf spot disease was significantly controlled by S2 cultured broth. Low sensitivity to several bactericides. S2 as a potential strain that is ready to process scale-up production and apply for integrated pest management.
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