Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89345
DC FieldValueLanguage
dc.contributorJenn-Wen Huangen_US
dc.contributor黃振文zh_TW
dc.contributor.authorPei-Hung Laien_US
dc.contributor.author賴佩鴻zh_TW
dc.contributor.other植物病理學系所zh_TW
dc.date2015zh_TW
dc.date.accessioned2015-12-07T07:25:33Z-
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dc.identifier.urihttp://hdl.handle.net/11455/89345-
dc.description.abstract乳酸菌廣泛分佈於自然界中,為一群可發酵六碳醣產生乳酸的桿狀或球狀細菌,屬於革蘭氏陽性菌,厭氧或兼性厭氧,不具泳動性,少數具有芽孢及莢膜,可有陰性觸酶反應之特性。乳酸菌能產生乳酸、乙酸、抗生素、細菌素、過氧化氫等抗生物質。市面上供作益生菌的乳酸菌有Lactobacillus, Bifidobacterium及Lactococcus等。文獻指出乳酸菌可預防Penicillium spp., Aspergillus spp., Fusarium spp.及Alternaria spp.等引起食物腐敗;亦有學者應用乳酸菌防治植物病原真菌,例如番茄萎凋病菌Fusarium oxysporum f.sp lycopersici與甜椒炭疽病菌Colletotrichum capsici等。近年來環保意識抬頭,開發友善環境及對人畜安全的生物製劑,用於防治植物病害是植病學者努力的工作目標,因此本研究的主要目的在於探討乳酸菌用於防治甘藍黑斑病 (由Alternaria brassisicola引起) 的可行性。首先由市售泡菜、優格、乳酸菌機能性食品分離獲得的細菌共計有27菌株,以及中興大學營養生理研究室提供Lactobacillus acidophilus LAP5, Lactobacillus acidophilus P2, Lactobacillus salivarius LS及 Lactobacillus farciminis S215等4乳酸菌株,利用de Man-Rogosa-Sharpe media (MRS) 培養基進行培養5天後,取它們的發酵液進行評估對於A. brassisicola ABA31分生孢子發芽的影響,結果發現LA21、S215及LS等菌株之發酵液可抑制病原菌的發芽率達90%以上。以甘藍切離葉生物分析法評估乳酸菌防治黑斑病的功效,結果發現LA21、S215及LS菌株之2倍MRS發酵稀釋液對於甘藍黑斑病的抑制率分別為100、95.5、80.9%。利用16S rDNA鑑定並在NCBI資料庫比對序列,將LA21鑑定為Lactobacillus helveticus。為了提升LA21、S215及LS菌株之防病能力及降低發酵成本,本研究經過系列評選測試菌株之培養基配方,結果發現以MRS-trehalose培養LA21菌株、MRS-glucose培養S215菌株及MRS-cellobiose培養LS菌株之8倍稀釋液後分別抑制A. brassisicola ABA31的分生孢子發芽率為95.7、80.7及87.3%。進一步,利用切離葉接種法評估乳酸菌發酵液加入1%CN多醣體對甘藍黑斑病的發生的影響,其中LA21-CN, S215-CN及LS-CN的500倍發酵稀釋液均可完全抑制黑斑病的發生外,尚可100%抑制病原菌的產孢。在溫室防治試驗中,甘藍植株澆灌10倍稀釋之LA21與S215發酵液後,它們抑制黑斑病的發生率分別為4.2%及14.5%。澆灌250倍稀釋之LA21-CN及S215-CN抑制黑斑病發生率分別為12.2%及46.9%。此外,噴灑20倍稀釋之LA21及S215發酵液抑制黑斑病發生率分別為58.8%與35.3%;噴灑250倍稀釋之LA21-CN與S215-CN抑制甘藍黑斑病發生率分別為100%及66.3%。在顯微鏡下觀察乳酸菌Lactobacillus helveticus LA21發酵液對甘藍黑斑病菌分生孢子形態之影響,發現10倍乳酸菌發酵稀釋液處理之孢子呈現原生質凝聚成顆粒狀;至於LA21-CN則造成孢子外壁厚薄不均,似有原生質滲漏的現象。進一步,評估乳酸菌發酵液衍伸應用之防治效果,發現稀釋10倍之LA21發酵液可抑制萵苣黑斑病菌Alternaria lactucae AL1-2及十字花科炭疽病菌Colletotrichum higginsianum PA01之孢子發芽;稀釋250倍之LA21-CN則可抑制蓮霧果腐病菌Pestalotiopsis eugeniae PW9、番石榴瘡痂病菌Pestalotiopsis psidii PG62、十字花科炭疽病菌PA01、萵苣黑斑病菌AL1-2、甘藍黃葉病菌Fusarium oxysporum f. sp. conglutinans Focn20及柑橘綠黴病菌Penicillium digitatum Cnp02之孢子發芽。利用乳酸菌發酵液防治細菌性軟腐病之效果評估,發現LA21及 LA21-CN發酵液均可抑制馬鈴薯與蘿蔔細菌性軟腐病的發生。綜合上述結果顯示乳酸菌Lactobacillus helveticus LA21及Lactobacillus farciminis S215發酵液分別添加CN多醣體後,兩者具有潛力發展成為防治植物病害之微生物源植保製劑。zh_TW
dc.description.abstractLactic acid bacteria (LAB) are a group of Gram-positive, acid-tolerant bacteria, sharing a number of metabolic and physiological characteristics. They are catalase-negative, non-motile, non-respiring and nonspore-forming rod-shaped bacteria or cocci. LABs are able to produce a wide range of antimicrobial compounds such as lactic acid, acetic acid, probionic acid, antibiotics, bacteriocins, and hydrogen peroxide. Lactobacillus, Bifidobacterium, and Lactococcus are commonly used as probiotics for humans and animals. LABs have also been shown to effectively suppress Penicillium spp., Aspergillus spp., Fusarium spp., and Alternaria spp. and are often used as the preservation agents for protecting food and feeds from decay. LAB culture broths have recently been demonstrated to be effective for controlling tomato Fusarium wilt caused by Fusarium oxysporum f.sp lycopersici and chilli anthracnose caused by Colletotrichum capsici. The purpose of this study was to evaluate the efficacy of LAB on controlling cabbage black spot disease caused by Alternaria brassisicola. Twenty seven strains of bactria were isolated from pickled vegetable, yogurt, commercially functional foods. In addition, there were also four strains of LAB, Lactobacillus acidophilus LAP5, Lactobacillus acidophilus P2, Lactobacillus salivarius LS and Lactobacillus farciminis S215, provided by the laboratory of Nutrition Physiology, Department of Animal Science, National Chung-Hsing University. The culture broths of LAB strains, LA21, S215, and LS grown in de Man-Rogosa-Sharpe (MRS) media for 5 days were able to inhibit over 90% conidial germination of A. brassisicola ABA31. The culture broths of LA21, S215, and LS at two-fold dilution were also effective respectively in reducing 100, 95.5 and 80.9% disease incidence of Alternaria black spot onto detached cabbage leaves. Based on the result of the sequences of partial 16S rDNA compared to NCBI database, LA21 was identified as Lactobacillus helveticus. It was proved that antagonistic ability of LABs to A. brassisicola ABA31 could be markedly influenced by various carbon sources in MRS medium used for culturing LABs. Among 17 carbon sources, trehalose, glucose, and cellobiose were respectively more suitable for enhancing LA21, S215, and LS to inhibit conidial germination of A. brassisicola ABA31. When the pathogen was treated with each culture broth of LA21 in MRS-trehalose, S215 in MRS-glucose, and LS in MRS-cellobiose at 8-fold dilution, its conidial germination was respectively reduced by 95.7, 80.7 and 87.3%. Furthermore, the culture broths of LA21, S215, and LS amended with 1% (w/v) CN (referred to LA21-CN, S215-CN, and LS-CN) at 500-fold dilution significantly reduced 100% disease incidence of Alternaria black spot in detached cabbage leaves and also completely inhibit sporulation of the pathogen on the inoculated leaves. In the greenhouse test, spraying 250-fold diluted culture broth of LA21-CN and S215-CN to cabbage seedlings could reduce disease severity of Alternaria black spot by 100% and 66.3%. However, drenching culture broth of LA21-CN and S215-CN at the same dilution rate could only reduce the disease severity by 12.2% and 46.9%. The microscopic observation indicated that the culture broth of L. helveticus LA21 could induce conidial protoplasm of A. brassisicola ABA31 aggregated and particulated, however LA21-CN culture broth did make cell wall of the conidia broken and appear protoplasm leakage. In advance, the study was extended to evaluate the effective spectra on plant protection. Results showed that 10-fold diluted culture broth of LA21 was effective in inhibiting conidial germination of Alternaria lactucae AL1-2 and Colletotrichum higginsianum PA01 and the culture broth of LA21-CN at 250-fold dilution was effective in inhibiting conidial germination of Pestalotiopsis eugeniae PW9, Pestalotiopsis psidii PG62, C. higginsianum PA01, A. lactucae AL1-2, Fusarium oxysporum f. sp. conglutinans Focn20 and Penicillium digitatum Cnp02. Data also showed that the culture broth of LA21 and LA21-CN could significantly suppress the bacterial soft rot of potato and radish caused by Pectobacterium carotovorum subsp. carotovorum ZL1. Based on the above results, it suggested that the culture broths of Lactobacillus helveticus LA21 and Lactobacillus farciminis S215 amended with 1% (w/v) CN have potential to be developed into microbial agent for controlling plant diseases.en_US
dc.description.tableofcontents目次 中文摘要……………………………………………………………………………....I 英文摘要……………………………………………………………………………..III 目次……………………………………………………………………………..........VI 表次索引…………………………………………………………………………......IX 圖次索引………………………………………………………………………….......X 前言……………………………………………………………………………………1 材料與方法……………………………………………………………………………5 一、乳酸菌株之分離、初步鑑定與保存……………………………………….5 二、供試植株…………………………………………………………………….6 三、病原菌供試菌株…………………………………………….………………6 四、乳酸菌對甘藍黑斑病菌孢子發芽的影響……….…………………………6 五、菌株拮抗能力之比較……………………………….………………………7 六、碳源對乳酸菌抑菌功效的影響………….……………………………....….7 七、乳酸菌培養於添加Cysteine之Man-Rogosa-Sharpe media (MRS) 之發酵液對甘藍黑斑病菌孢子發芽的影響……………………………..………....7 八、LA21菌株之16S rDNA鑑定…………………………….............…..........8 (一) DNA萃取……………………………………………….............…......8 (二) 聚合酶連鎖反應 (Polymerase Chain Reaction) ……….............…….8 (三) 增幅片段之定序與比對………………………………..............…......8 九、不同濃度CN多醣體對甘藍黑斑病菌孢子發芽的影響……….……….......9 十、LA21, S215及LS發酵液加入1%CN多醣體對甘藍黑斑病菌孢子發芽的影響……………………….……………………………………………...…..9 十一、利用切離葉法評估乳酸菌發酵液及乳酸菌發酵液添加1%CN多醣體對甘藍黑斑病發生與病原菌產孢的影響……………………………….....….9 十二、溫室防治試驗…….…………………………………………….…......….10 (一) 澆灌處理法……………………………………………………..........10 (二) 噴霧處理法………………………………………………..............…11 十三、乳酸菌Lactobacillus helveticus LA21發酵液對甘藍黑斑病菌分生孢子形態之影響…………………………………………………..................…11 十四、乳酸菌發酵液應用於拮抗其他植物病原真菌孢子發芽之評估…....…12 十五、乳酸菌發酵液應用於防治細菌性軟腐病之效果評估.......................….12 結果……………………………………………………………….……….............…14 一、乳酸菌株之分離與鑑定…………………………….………...................…14 二、乳酸菌對甘藍黑斑病菌孢子發芽的影響………….…….…..................…14 三、菌株拮抗能力之比較……………………….………...............................…15 四、碳源對乳酸菌抑菌功效的影響…….……...............................................…15 五、乳酸菌培養於添加Cysteine之Man-Rogosa-Sharpe media (MRS) 之發酵液對甘藍黑斑病菌孢子發芽的影響………............................................…15 六、不同濃度CN多醣體對甘藍黑斑病菌孢子發芽的影響..........................…16 七、LA21, S215及LS發酵液加入1%CN多醣體對甘藍黑斑病孢子發芽的影響……………….………...........................................................................…16 八、利用切離葉法評估乳酸菌發酵液及乳酸菌發酵液添加1%CN多醣體對甘藍黑斑病發生的影響…............................................................................…16 九、利用切離葉法評估乳酸菌發酵液及乳酸菌發酵液添加1%CN多醣體對甘藍黑斑病菌產孢之影響.......................................................................….…17 十、溫室防治試驗......................................................................................…..…18 (一) 澆灌處理法......................................................................................….…18 (二) 噴霧處理法......................................................................................….…18 十一、乳酸菌Lactobacillus helveticus LA21發酵液對甘藍黑斑病菌分生孢子形態之影響...........................................................................................….…18 十二、乳酸菌發酵液應用於拮抗其他植物病原真菌之效果評估.................…19 十三、乳酸菌發酵液應用於防治細菌性軟腐病之評估.........................…...….19 討論……………………………………………………………….……….............…21 參考文獻……………………………………………………….……….................…26 圖表……………………………………………………………….……….............…32 附錄……………………………………………………………….……….............…58 表次索引 表一、三十一株細菌菌株於Man-Rogosa-Sharpe media (MRS) 培養之菌液對甘藍黑斑病菌Alternaria brassisicola ABA31分生孢子發芽之抑制效果。...........32 表二、以17種碳源的Man-Rogosa-Sharpe media (MRS) 各別培養LA21, S215及LS菌株五日之發酵液對甘藍黑斑病菌Alternaria brassisicola ABA31分生孢子發芽之影響。.....................................………………….………...................33 表三、以九種不同碳源的Man-Rogosa-Sharpe media(MRS)培養LA21, S215及LS菌株五日之不同濃度發酵液對甘藍黑斑病菌分生孢子發芽之影響。……...34 表四、不同濃度的LA21, S215及LS發酵液對於甘藍黑斑病菌在甘藍切離葉上產孢之影響。……………………………………………………………………..35 表五、不同濃度CN多醣體對甘藍黑斑病菌Alternaria brassisicola ABA31孢子發芽之影響。……………………………………………………………………...36 表六、添加1%CN多醣體的LA21, S215及LS發酵液之不同濃度稀釋液對於黑斑病在甘藍切離葉產孢之影響。……………………………..……………...37 表七、溫室中評估Lactobacillus helveticus LA21及Lactobacillus farciminis S215發酵液稀釋10倍及20倍防治甘藍黑斑病之效果。…………………………38 表八、溫室中評估Lactobacillus helveticus LA21及Lactobacillus farciminis S215發酵液加入1%CN多醣體稀釋250及500倍防治甘藍黑斑病之效果。…....39 表九、Lactobacillus helveticus LA21發酵液對8種植物病原真菌孢子發芽之影響。………………………………………………………………….………....40 表十、Lactobacillus helveticus LA21發酵液對8種植物病原真菌孢子形態之影響。……………………………………………………………………….…....41 圖次索引 圖一、乳酸菌之MRS發酵液和菌體懸浮液對甘藍黑斑病菌Alternaria brassisicola ABA31分生孢子發芽之影響。……………………………..………….……….43 圖二、將LA21, S215及LS於最佳碳源MRS培養液添加0 %、0.05%及0.1% Cysteine培養5天,稀釋不同濃度對甘藍黑斑病菌分生孢子發芽之影響。….44 圖三、不同濃度的LA21, S215及LS發酵液對黑斑病在甘藍切離葉發生之影響。………………………………..………………………………………….….45 圖四、不同濃度的LA21, S215及LS發酵液對黑斑病在甘藍切離葉發生之影響。……………………………………..…………………………………….….46 圖五、添加1%CN多醣體的LA21, S215及LS發酵液之不同濃度稀釋液抑制甘藍黑斑病菌Alternaria brassisicola ABA31孢子發芽之效果。……………….47 圖六、添加1%CN多醣體的LA21, S215及LS發酵液之不同濃度稀釋液對於黑斑病在甘藍切離葉發生之影響。……………………..……………………….48 圖七、添加1%CN多醣體的LA21, S215及LS發酵液之不同濃度稀釋液對於黑斑病在甘藍切離葉發生之影響。………………………………………………49 圖八、十字花科黑斑病標準面積圖示。……………………………………………50 圖九、在溫室內評估添加1%CN多醣體之Lactobacillus farciminis S215及Lactobacillus helveticus LA21發酵液防治甘藍黑斑病之效果。………….….51 圖十、在溫室中評估添加1%CN多醣體之Lactobacillus farciminis S215發酵液稀釋250倍防治甘藍黑斑病之效果。…………………………………….………52 圖十一、在顯微鏡下觀察Lactobacillus helveticus LA21發酵液對甘藍黑斑病菌Alternaria brassisicola ABA1分生孢子形態之影響。…………………..….…53 圖十二、Lactobacillus helveticus LA21發酵液抑制Pectobacterium carotovorum subsp. carotovorum引起馬鈴薯軟腐病的效果。………………………………54 圖十三、Lactobacillus helveticus LA21發酵液抑制Pectobacterium carotovorum subsp. carotovorum引起馬鈴薯軟腐病的效果。………………………..….…55 圖十四、Lactobacillus helveticus LA21發酵液抑制Pectobacterium carotovorum subsp. carotovorum引起蘿蔔軟腐病的效果。…….…………………………..56 圖十五、Lactobacillus helveticus LA21發酵液抑制Pectobacterium carotovorum subsp. carotovorum引起蘿蔔軟腐病的效果。…….…………………………..57zh_TW
dc.language.isozh_TWzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2018-08-28起公開。zh_TW
dc.subject乳酸菌zh_TW
dc.subject生物製劑zh_TW
dc.subject甘藍黑斑病菌zh_TW
dc.subject細菌性軟腐病菌zh_TW
dc.subjectLactic acid bacteriaen_US
dc.subjectbiocontrol agenten_US
dc.subjectAlternaria brassisicolaen_US
dc.subjectPectobacterium carotovorum subsp. carotovorumen_US
dc.title研製乳酸菌植保製劑防治甘藍黑斑病zh_TW
dc.titleEvaluation for the Efficacy of Lactic Acid Bacteria on Controlling Alternaria Black Spot Disease of Cabbageen_US
dc.typeThesis and Dissertationen_US
dc.date.paperformatopenaccess2018-08-28zh_TW
dc.date.openaccess2018-08-28-
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