Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31787
標題: 以鏈黴菌Streptomyces fimbriatus WRS9作為生物製劑之潛力及鏈黴菌屬聚合酶連鎖反應專一檢測引子之發展
The potential of Streptomyces fimbriatus WRS9 as a microbial biofungicide and the development of polymerase chain reaction primer for specific detection of Streptomyces spp.
作者: 邱瓊瑩
Ying, Chiu Chiung
關鍵字: Streptomyces spp.;鏈黴菌;microbial biofungicide;polymerase chain reaction;PCR;生物製劑;聚合酶;連鎖反應
出版社: 植物病理學系
摘要: 
陸、中文摘要
以鏈黴菌Streptomyces fimbriatus WRS9作為生物製劑之潛力及鏈黴菌屬聚合酶連鎖反應專一檢測引子之發展
邱瓊瑩
本研究在探討Streptomyces fimbriatus WRS9與S. griseobrunneus WRS3、S. lincolnensis WRS7與S. saraceticus SS31等菌株比較其相關的生物特性後,評估WRS9其應用作為防治植物病害的生物製劑的潛力。WRS9是自甜菜根圈土壤中所分離得到的菌株,在初步的篩選測試中,其幾丁質的分解能力以及對稻熱病菌 (Pyricularia oryzae)、腐霉病菌 (Pythium aphanidermatum) 與木瓜疫病菌 (Phytophthora palmivora) 的拮抗能力均較其他三個菌株來的強。在搖瓶系統當中,以膠狀幾丁質 (1% chitin) 與Rhizoctonia solani AG4之菌絲懸浮液做為培養基質均可誘導WRS9產生至少6條同功異構的幾丁質分解酵素,分子量大小約在32-60 kDa之間。WRS9在2-3% 燕麥培養液中生長良好,接種後第6天生質體產量可達3.19x108 cfu/ml,相較之下其在2% 玉米與2% 粉頭培養液生長較差,生質體產量在接種6天後只有2.03-1.99x107 cfu/ml,相同於菌的生長,燕麥與玉米的培養濾液對P. aphanidermatum的拮抗效果自接種第一天開始可持續至第五天,待第六天後才無法檢測到抗生活性,然而在粉頭培養濾液中,除了在第五天可檢測到微弱的抗生活性之外,在接種後的六天當中,幾乎沒有抗生活性的表現。從7種天然培養基中篩選對WRS9具有最佳產孢效果的培養基,其中以添加0.2% 麥芽抽出物的馬鈴薯蔗糖培養基 (potato-sucrose-malt extract, PSMe) 的效果最好,在培養第五天後孢子產量可達4.67x109 cfu/plate,其他供試的培養基包括粉頭、PSA與幾丁質平板對WRS9也具有不錯的產孢效果,孢子產量可達2.78-3.36x109 cfu/plate,將上述來自不同培養基平板的孢子懸浮液製成接種源而接種至燕麥培養液後,對於WRS9的生質體產量除了來自2% 玉米平板的接種源之外,其餘的接種源彼此間效果相當。在供試菌的先導量產方面,藉由過去在SS31所建立並在WRS3所驗證確具良好效果的量產技術流程,嘗試以5 L與50 L攪拌式發酵槽進行試驗,但所獲得的生質體產量仍是維持在108 cfu/ml以下。雖然量產條件有待細部的調整,但仍以木瓜根腐病 (由Phytophthora palmivora所引起) 作為試驗對象,評估施用WRS9液態培養液對病害防治的潛力,在PSB中培養了7天的P. palmivora菌絲懸浮液,接種3x106 cfu/ml 的WRS9孢子懸浮液或燕麥培養液,可在接種後接近24-36小時測得有電解質滲漏增加的情形產生,明白的顯示出其對於細胞膜的抗生作用,在光學顯微鏡的檢視下,更進一步觀察到病原菌的菌絲會被WRS9的菌絲所穿生與纏繞,使得菌絲失去其細胞組成份且整個菌絲結構被拆解或甚至是完全被瓦解,此種對於病原菌菌絲的超寄生與結構性的破壞,似乎是意味著WRS9水解酵素的功能。在溫室試驗中,澆灌500至1000倍稀釋的WRS9培養液可約略降低木瓜幼苗受到人工接種P. palmivora的感染,然而澆灌100倍稀釋的培養液卻會嚴重降低幼苗的存活率,推測對幼苗有此一危害的影響,可能的原因乃是由於抗生物質對於根系造成的毒害。在木瓜果實上人工接種P. palmivora菌絲盤塊,並在接種前一天施用WRS9培養液105-106 cfu/ml可延遲或抑制P. palmivora菌絲在果實上的感染。
本研究的另一工作是發展專一性引子對以聚合酶連鎖反應來檢測與鑑定鏈黴菌屬的細菌。此研究的主要目的在於監控連續施用拮抗性鏈黴菌株的生質體於病害防治應用上對於生態環境可能造成的衝擊提供一有效的工具。抽取來自WRS2、WRS3、WRS7、WRS9、TYS15、TYS17、TYS19、SS31與WRS38等9個鏈黴菌菌株的基因體DNAs,並以適用性引子對 (UNI 16S-L與UNI 16S-R) 增幅其16S rDNAs並選殖到yT&A載體內,所有菌株選殖的16S rDNAs片段大小約為1400 bp,而WRS3、WRS7、WRS9與SS31所選殖的16S rDNA基因已完成解序的工作,利用國家衛生研究院生物資訊網所提供的Pretty分析軟體 (Seq-Web) 與NCBI (National Center for Biotechnology Information) 資料庫進行線上比對以及序列分析,結果顯示選殖的WRS7 16S rDNA序列和資料庫內的Streptomyces sp. 16S rDNA 基因均具有95% 以上的相同度。比較已解序的四個菌株間的序列更可發現序列中存在著特定具有種專一性 (species-specific) 的區域位於第171-181鹼基中,因此根據此一結果分別自各菌株的171-181鹼基中設計正向 (forward) 的引子WRS3-L、WRS9-L、WRS7-L與SS31-L,並設計2個位於核甘酸序列679-697與951-968的反向 (reverse) 引子ScR-1與ScR-2。嘗試利用WRS3-L、WRS7-L與WRS9-L作為專一性的正向引子,UNI 16S-R、ScR-1與ScR-2作為反向引子,分別以S. griseobrunneus WRS3、S. lincolnensis WRS7、S. fimbriatus WRS9、S. saraceticus SS31、其他5個Streptomyces spp.菌株WRS2、WRS38、TYS15、TYS17與TYS19以及其他5個非鏈黴菌類的細菌,包括了Bacillus sp.、Pseudomonas sp.、Erwinia sp.、Xanthomonas sp. 1和2與Ralstonia sp.等的基因體DNA作為模板進行PCR檢測,結果顯示以WRS3-L/ScR-2作為引子對進行PCR反應只有WRS3菌株可增幅出預期的797 bp.大小之專一性片段,以WRS7-L/ScR-2作為引子對進行PCR反應只有WRS7菌株可增幅出預期的797 bp.大小之專一性片段,以WRS9-L/ScR-2作為引子對進行PCR反應只有WRS9菌株可增幅出預期的797 bp.大小之專一性片段。相反的,其他正向與反向引子對的配合並無法專一性地檢測出欲偵測的鏈黴菌菌株。在檢測WRS9的專一性PCR反應方面,利用系列稀釋的WRS9孢子懸浮液取代基因體DNA作為模板測試其引子對的靈敏度,結果發現,以WRS9-L/UNI 16S-R作為引子對時,可成功的偵測出預期的1266 bp的片段且偵測的下限濃度可為將108 cfu/ml稀釋至10-4倍。為了檢測與鑑定專一的鏈黴菌的種與菌株,在偵測特定菌株的詳細偵測方法須進一步的改進,但使用16S rDNA上特殊序列作為正向的引子仍不失為一有價值的方法。

柒、英文摘要
The potential of Streptomyces fimbriatus WRS9 as a microbial biofungicide and the development of polymerase chain reaction primer for specific detection of Streptomyces spp.
Chiung-ying Chiu
In the discussed thesis works, the biological characteristics of Streptomyces fimbriatus WRS9 pertaining to its potential application as a biofungicide for plant disease control were evaluated using S. griseobrunneus WRS3, S. lincolnensis WRS7, and S. saraceticus SS31 as compared strains. In a preliminary screening test, WRS9-which was originally isolated from sugarcane rhizosphere, was found superior in strength of chitinolytic activity and antagonisity against Pyricularia oryzae, Pythium aphanidermatum and Phytophthora palmivora, than that of the three compared strains. In a broth culture system under continuous shaking, it produced at least 6 inducible chitinase isozymes with molecular weight ranging from 32 to 60 kDa, in responding to the presence of colloidal chitin or the mycelial suspension of Rhizoctonia solani AG4. The bacterium grows well in 2-3% oat broths, the biomass yield reached 3.19x108 cfu/ml 6 days after inoculation (DAI). As a comparison, the bacterial growth in 2% corn and 2% wheat bran broth were comparatively less well; the biomass yield reached only 2.03-1.99x107 cfu/ml at six DAI. In paralleled to the bacterial growth, the antagonisity against P. aphanidermatum was detected from all oat and corn broth cultures at 1 DAI which then hold throughout the 5th day after inoculation and declined to non-detectable at 6 DAI . In a wheat bran broth culture, however, the antagonisity remained non-detectable throughout the 6 days culture period except that a slight increase of antagonisity was detected at 5 DAI. A total of 7 natural agar media were screened for the performance for supporting the sporulation of the test bacterium. Among them, the 0.2 % maltextract amended potato sucrose agar (PSMe) appeared to be the best, the spore yield reached approximately 4.67x109 cfu/plate at 5 DAI. Other test media with good performance in supporting the sporulation of WRS3 include 2% wheat bran, PSA and chitin agar; the yield of biomass all achieved 2.78-3.36x109 cfu/plate level. When applied as inoculum for liquid culture using oat broth medium, spore samples obtained from all these natural agar media all performed equally well in regarding to the yield of biomass except that from 2% corn agar which tended to have a lower yield. A pilot scale mass production of test bacterium was attempted using 5 to 50 L stirrer type fermentor. By an established protocol developed for SS31 and as well for WRS3, the yield of biomass obtained all remained less than 108 cfu/ml. Although the fine tune of the protocol need to be worked out, the potential application of the broth cultures produced in disease control was attempted using papaya root rot (caused by Phytophthora palmivora) as trial target. In a 7-day old suspension culture of P. palmivora growing in PSB broth, the inoculation by WRS9 at 3x106 cfu/ml caused an increased leakage of electrolytes approximately 24 to 36 hours after inoculation; an antibiotic function on the membrane damage was clearly indicated. A followed examination by light microscopy further revealed that the pathogen mycelia were penetrated and/or tangled up by WRS9 mycelium, lost their cellular contents and were finally totally dismantled or even disintegrated. The mycoparasitic and structural damaging effect on pathogen mycelium seemed to implicate the functioning of lytic enzymes of WRS9. In a greenhouse trial, the drenching of 500 to 1000X diluted WRS9 cultural broth was shown slightly reduced the infection of papaya seedlings by artificially inoculated P. palmivora. The application of WRS9 cultural broth at 100X dilution however, greatly reduced the survival of the seedlings. A plausible reason for the observed deleterious effect on test seedlings appeared to due mainly the antibiotic toxicity on the root system. On papaya fruit artificially inoculated with P. palmivora zoospore suspension, the application of WRS9 at 105-106 cfu/ml in concentration one day before inoculation significantly deterred or even stopped the fruit infection.
Another focus of this thesis work was the development of primer for specific detection and/or identification by polymerase chain reaction the members of Streptomyces spp. The major objective of the devoted efforts was to provide useful tool for monitoring the possible ecological impacts due to repeated application of viable propagules of the antagonistic Streptomyces strains attempted for disease control application. The genomic DNAs were extracted from a total of 9 Streptomyces strains namely WRS2, WRS3, WRS7, WRS9, WRS38, TYS15, TYS17 TYS19 and SS31. And from each of them, the 16S rDNAs were amplified by PCR using universal primer pair UNI16S-L and UNI16S-R and cloned by yT&A vector. All the cloned 16S rDNAs appeared to be approximately 1400 bps in size; the full sequence of that from WRS3, WRS7, WRS9 and SS31 were resolved. The sequence analysis by Pretty software (Seq-Web) using database from NCBI indicated that the cloned sequences from WRS7 had 95% identities comparing to that known for most Streptomyces. The comparative analysis of the 4 resolved sequences further indicated the existence of certain species-specific regions located at 171-181 respectively. And from these data information, WRS3-L, WRS7-L, WRS9-L and SS31-L each respectively as forward primer specific for the denoted strain were designed from 171-181 region; and likewise ScR-1 (679-691) and ScR-2 (951-968) were each designed to serve as the reverse primer. The followed extensive PCR amplification trial using WRS3-L, WRS7-L, WRS9-L each as specific forward primer, 16S-R, ScR-1, and ScR-2 each as reverse primer, and the genomic DNAs obtained from S. griseobrunneus WRS3, S. lincolnensis WRS7, S. fimbriatus WRS9, 5 other Streptomyces spp. including WRS2, WRS38, TYS15, TYS17, and TYS19, and 5 other non-Streptomyces bacteria including Bacillus sp., Pseudomonas sp., Erwinia sp., and Xanthomonas spp. 1 and 2, each respectively, as template DNA. The results obtained indicated the use of WRS3-L/ScR-2 as primer pair detected the predicted 797 bps fragment only from WRS3; that of WRS7-L/ScR-2 detected the 797 bps fragment only from WRS7; whereas that of WRS9-L/ScR-2 detected the 797 bps fragment only from WRS9. All the other tested forward/reverse primer pair combinations, as the contrary, were not successful in terms of specific detection of the attempted bacterial strain. For the specific PCR detection of WRS9, the spore suspension at serial dilution was tested for its efficacy in replacing the template function of genomic DNAs. The application of WRS9-L/UNI 16S-R as primer pair was successful in detecting the predicted 1266 bps fragment; the detection limit went down to about 108 cfu/ml diluted 10-4 level. For the detection/identification of specific Streptomyces species/strains, the detailed protocol of the detection may need further improvement for each specific target strain, the use of species specific 16S rDNA sequence as forward primer appeared to be a valuable tool.
URI: http://hdl.handle.net/11455/31787
Appears in Collections:植物病理學系

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