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標題: Streptomyces sp. strain S1抗生物質之產生及其於卵菌綱病原危害防治之作用機制初探
A preliminary study on the production of antibiotic by Streptomyces sp. strain S1 and the possible mechanism it may contribute to the control of diseases caused by oomycetes
作者: 林佳蓉
Lin, Chia-Jung
關鍵字: Streptomyces;鏈黴菌;antibiotics;oomycetes;mechanism;抗生素;卵菌綱;作用機制
出版社: 植物病理學系所
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供試鏈黴菌Streptomyces sp. S1菌株為賴氏 (2003) 由市售栽培用泥碳土材料分離獲得,先前研究中已證實其在土壤傳播性真菌或類真菌病原防治用生物殺菌劑發展上確具應用潛力。有關其病害防治應用效果也證實與其優異幾丁質酶與葡萄聚醣分解酶活性所附予之超寄生性、以及所產生二次代謝物的廣效性抗生性有密切關係。本研究旨在針對素具惡名且於世界各地常見造成作物生產嚴重損失之卵菌綱植物病原菌,瞭解此一供試菌株抗生性之作用機制,以為未來推廣應用之參考。S1菌株經於搖瓶系統以可溶性澱粉作為取代性碳源之Czapek’s修改配方培養液行震盪培養,於培養6天後所收取培養濾液,利用對峙培養生物活性測試,本研究證實其對荔枝露疫病菌 (Peronophythora litchii)、番椒疫病菌 (Phytophthora capsici) 與柑橘裾腐病 (Phytophthora palmivora) 等病原卵菌之菌絲生長均有優異的菌絲生長抑制效果,且其抑制性與應用劑量明顯有關。為進一步了解所見抗生性之分子作用基礎,試驗中嘗試利用乙酸乙酯萃取抗生物質成分,存在於有機層之萃取物繼之經減壓濃縮至乾,再以甲醇定量回溶,所獲得抗生物質粗萃取液同上述對峙培養方法測試,發現其抗生活性表現與測試病原種類攸關;以供試卵菌綱之類真菌 (fungal like) 病原菌為例,其對荔枝露疫病菌和柑橘裾腐病菌的抑制效果即明顯優於番椒疫病菌P. capsici及甘藍幼苗猝倒病菌Pythium aphanidermatum,而於常見真菌病原之供試菌株,按敏感性依次為草莓炭疽病菌Colletotrichum gloeosporioides、立枯絲核菌Rhizoctonia solani AG4、甘藍黑斑病菌Alternaria brassicae以及草莓灰黴病菌Botrytis cinerea,然對胡瓜萎凋病菌Fusarium oxysporum f. sp. cucumerinum之菌絲生長則看不出明顯抑制效果,此外其對蓮霧青枯病菌Ralstonia solanacearum、軟腐病菌Erwinia carotovora subsp. carotovora 與 Erwinia chrysanthemi、柑橘潰瘍病菌Xanthomonas axonopodis pv. citri及芒果黑斑病菌Xanthomonas campestris pv. mangiferaeindicae等細菌性病原之菌落生長亦全無抑制作用;另以荔枝露疫菌、草莓炭疽病菌及灰黴病菌為供試材料則發現,所獲得之抗生物質粗萃取液對其孢囊與分生孢子之發芽均有明顯之抑制效果,其主要影響包括發芽延遲、發芽管生長遲緩及嚴重畸形發育,於供試抗生物質影響下發芽管多呈粗短狀、分枝頻率高之簇生狀,有些更於末端異常膨大形成短肥畸形如同芽點一般大小的側枝。值得注意的是此發芽管畸形發育另伴隨著附著器無法形成;本研究亦證實此一對荔枝露疫病菌孢囊發芽及發芽管生長發育之抑制作用在生體外(玻片培養)及生體內(接種於荔枝葉片)系統均可同樣發揮效果,荔枝葉片接種試驗更已證實,以供試S1之抗生物質粗萃取液添加處理可抑制孢囊正常發芽及附著器形成,進而成功抑制露疫病菌之感染能力。為進一步瞭解此些抑制活性可能之作用機制,本研究同樣以荔枝露疫病菌為測試材料,利用抗生物質粗萃取液系列探討其對細胞膜通透性、能量代謝以及細胞壁生合成與堆置作用等影響,經以螢光染劑fluorescent brightener 28添加檢視供試菌細胞壁組成,於共軛焦螢光顯微鏡輔助下,證實經抗生物質粗萃取液處理後,發芽管與新生菌體正常之細胞堆置作用均已被抑制。同一試驗處理,另以掃描式電子顯微鏡高倍檢視則發現,於發芽菌絲延伸生長部位可見有明顯菌絲體縊縮情形,且其末端則可見多數意圖分枝生長未成功而呈明顯膨大之構造。繼而以電導度測定儀檢測供試抗生物質粗萃取液添加處理對荔枝露疫病菌菌絲導電度變化,試驗結果證實其影響在處理8小時後即明顯快速增加,顯示抗生物質處理對供試菌細胞膜完整性與通透性均有明顯的破壞作用;另參考Cox試驗流程,以alamarBlue®染劑做為分子探針,檢測供試抗生物質粗萃取液添加處理對荔枝露疫病菌孢囊與能量代謝攸關的還原力 (reducing power) 之影響,螢光反應結果證實,其對還原力之降低效果與市售QiI類呼吸作用抑制性殺菌劑安美速 (Amisulbrom) 之效果相近,此是否顯示本研究所檢測之供試抗生物質與安美速具有同樣的作用機制值得繼續探討。接續為瞭解此抗生物質之化學特性,S1粗萃取液經以Sep-Pak C18® cartridge去雜淨化,配合對P. litchii、Ph. palmivora及Pythium aphanidermatum等病原卵菌之拮抗活性檢測,證實以80%甲醇沖提回收之樣品為主要含抗生物質之區段,進一步利用逆相式高效液相層析系統全光譜掃描,初步得知S1抗生物質於251nm波長下具最大吸收值,雖抗生物質之純化工作仍有待完成,本研究中所提供證據已明白指出,此一標靶抗生物質對廣譜域經濟重要性真菌與類真菌病原咸具優異抗生活性,生理性影響測試亦已證實其於孢囊或分生孢子發芽所造成之抑制作用與導致畸形生長效果,一再顯示其與習知化學藥劑常見對細胞膜通透性之影響、對細胞壁堆置作用之影響、及對能量代謝之影響密切相關,更增添此標靶抗生物質作為實際應用植物保護劑之可行性,有關此抗生物質之純化與分子結構及病害防治作用機制之闡明將為本研究未來繼續努力之重點。

The antagonistic Streptomyces sp. strain S1 isolated from commercial peat moss (Lai, 2003) was shown of great potential as a microbial bio-fungicide for the control of plant diseases- especially those caused by soil borne fungal/fungal-like pathogens. The effectiveness of disease control that due greatly in part to the mycoparasitic effect because of its excellence in chitinase and glucanase activities, and as well the antagonistic activity resulted from the production of antibiotic metabolites had been demonstrated. The main objective of this study was to explore the mechanism contributing to the antagonistic effectiveness of this test strain against plant pathogenic oomycetes, one of the most notorious pathogens causing tremendous yield losses in crop production worldwide. For the production of antibiotic(s), the test strain S1 was cultured in a modified Czapek's broth wherein soluble starch was applied as a substitute for sucrose. By dual culture bioassay, the cultural filtrates obtained from 6-day old broth culture were shown to be inhibitory on the mycelial growth of Peronophythora litchii, Phytophthora capsici, and Phytophthora palmivora. The observed inhibitory effect appeared to be dose dependent. To explore the molecular basis of the antagonistic effectiveness, extraction of the antibiotics was attempted by use of ethyl acetate, the organic phase obtained was flash evaporated to dryness, and the crude extract obtained was re-dissolved by methanol. By similar dual culture assay, the inhibitory effect of the crude extract was found to be dependent on sensitivity of pathogen strains. For oomycete pathogens tested, the antibiotic effectiveness was better for P. litchii and Ph. palmivora as compared to that for Ph. capsici and Pythium aphanidermatum. Likewise, for fungal pathogens tested, the extent of growth inhibition of the tested fungal strains appeared to be in the order Colletotrichum gloeosporioides, Rhizoctonia solani AG4, Alternaria brassicae and Botrytis cinerea. For Fusarium oxysporum f. sp. cucumerinum, however, the inhibitory effect on mycelial growth was not detected. The lack of inhibitory effect was also observed on the bacterial pathogens including Xanthomonas axonopodis pv. citri, Xanthomonas campestris pv. mangiferaeindicae, Erwinia carotovora subsp. carotovora, Erwinia chrysanthemi and Ralstonia solanacearum. In addition, the application of S1 antibiotic crude extract was also found to be inhibitory on the sporangial germination of P. litchii, and conidial germination of C. gloeosporioides and B. cinerea. The inhibitory effect was manifested by the greatly delayed germination, the significantly deterred and extensively malformed germ tube development. The affected germ tubes were characterized by stunted growth, highly increased branching and/or formation of short lateral buds. It was also worth noting that the germ tube malformation was apparently associated with the incapability of appressorial formation. For P. litchii, the inhibitory effect on sporangial germination and germ tube development was demonstrated by both in vitro (on glass slides) as well as in vivo (on inoculated litchi leaves) systems. On litchi leaves, the application of S1 antibiotic crude extract was shown successfully inhibited the normal sporangial germination and appressorial formation, and thus the infection of P. litchii. In order to learn the mode of action of these observed inhibitory activities, the effect of S1 antibiotic crude extract on cell wall synthesis and deposition, membrane permeability, and energy metabolism of P. litchii were investigated. By application of fluorescent brightener 28 to reveal the cell wall constituents, results obtained by confocal fluorescent microscopy clearly indicated the lack of deposition of normal cell wall constituents on the germ tubes and new mycelial growth on the S1 antibiotic treated sporangia or mycelia. The morphogenetic impact was further shown by an adjunct scanning electron microscopy where that constriction of mycelial extension and failure of germ tube and branching bud growth were observed. Upon antibiotic addition, an increased electrolyte leakage was detected from the test samples 8 hours after treatment. The deteriorative or even disruptive effect of S1 antibiotic crude extract on membrane permeability was clearly indicated. Furthermore, with the addition of alamarBlue® as indicative molecular probe, the inhibitory effect of S1 antibiotic crude extract on the reduction potential of P. litchii sporangia comparable to that by amisulbrom treatment was demonstrated. Whether or not this suggested the effect of applied S1 crude extract on the energy metabolism a function similar to that by the QiI inhibitor chemical fungicides, however, remained to be further investigated. For chemical characterization of the antibiotic explored, the antibiotic containing crude extract was purified first by Sep-Pak C18® cartridge clarification with the use of 80% methanol as eluent. The antibiotic activity of the collected eluents was confirmed by bioassay using P. litchii, Ph. palmivora and Pythium aphanidermatum as test targets. Further purification of the antibiotic by reverse phase high performance liquid chromatography (HPLC) is now in process. Although the chemical characteristics of the antibiotic are awaited to be resolved, the data herein provided have pointed clearly its inhibitory effect against wide range spectrum of fungal/fungal like pathogens. And the physiological impacts demonstrated including effects on membrane permeability, wall deposition, and energy metabolism all explained well the morphogenetic abnormality observed in sporangial/spore germination and implicating the usefulness of the target antibiotic as plant protectant. The purification of the antibiotic and the elucidation of its mode of action relating to disease control will be the major focuses for our future endeavor.
其他識別: U0005-1808201116163100
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