臺灣博碩士論文加值系統

十字花科黑斑病，是由Alternaria brassicicola (Schw.) Wiltshire所引起，於世界各地之十字花科蔬菜作物普遍發生，其病原菌可影響寄主植物生長的所有階段，包括種子繁殖，雖有推薦化學藥劑供防治應用，然而藥劑殘留對人體與環境之淺潛在危險性，使開發替代性的微生物製劑為當務之急。本研究旨在探討本土拮抗性枯草桿菌TKS1-1對黑斑病防治之抗生物質鑑定及其可能作用機制。本研究由包括TKS1-1、SP4-17、WG6-14及WP8-12等4株研究室既有抗生活性與生長產孢性狀俱為優異之BSG菌株，經由對峙培養篩選，證實對十字花科黑斑病菌均具有良好的拮抗活性，且以枯草桿菌TKS1-1培養於PSB中5天之培養濾液對黑斑病菌具最佳拮抗能力。在離葉接種系統中，接種病原菌後一天施用TKS1-1培養濾液不具抑制感染能力；相反的，於接種病原菌同時與前一天施用TKS1-1培養濾液則可防治病原菌侵入感染。且TKS1-1培養濾液所含拮抗物質對溫度及pH具高度穩定性，於pH值2 - 13及100 ℃處理20分鐘仍具拮抗活性。其濾液於生物體外，主要影響包括發芽延遲與發芽管產生不正常膨大畸形結構，於生物體內，接種於甘藍葉片上均可發揮同樣效果，值得注意的是此發芽管畸形結構，使得類附著器結構無法形成；且其濾液影響菌絲細胞膜的通透性，對細胞膜造成傷害，使細胞膜內電解質流失產生離子滲漏，於處理4小時後顯著增加2.1倍，至28小時電解質滲漏達到高峰。為鑑定抗真菌物質之特性，試驗中以正丁醇、乙酸乙酯、Sep-pakR C18及酸沉降等萃取方式萃取，其中以正丁醇萃取具最佳拮抗能力，其萃取液經薄層色層分析板顯示於Rf 值0.06、0.15、0.36可偵測到具抗真菌活性，利用呈色反應證實所產生之抗真菌物質可能具有胺基酸及脂質等特性。另經Sep-pakR C18管柱吸附，以70 %甲醇溶液之沖洗液，進一步以高效液相層析儀分析其滯留時間於3.43及3.98分鐘之物質，其UV吸收光譜近似於iturin A混合標準品。進一步將高效液相層析儀分離之抗生物質，以三段四極桿串聯式質譜儀，分析其母離子Q1與子離子Q3片段，顯示於3.43分鐘的母離子Q1分子量為1043 m/z，其具b-type子離子片斷為212 m/z (b2)、299 m/z (b3)、524 m/z (b4)、638 m/z (b5)、801 m/z (b6)、915 m/z (b7) 等特徵離子片斷，故證實為iturin A2 (C14)；而3.98分鐘的母離子Q1分子量為1057 m/z，其具b-type子離子片斷為212 m/z (b2)、299 m/z (b3)、538 m/z (b4)、652 m/z (b5)、815 m/z (b6)、929 m/z (b7) 等特徵離子片斷，故證實為iturin A3,4,5 (C15)。且於過去研究中，已知iturins類抗生物質脂肪酸碳鏈愈長可增加其抗菌活性，亦有相同結果。本研究結果顯示枯草桿菌TKS1-1所產生的iturin A是抑制A. brassicicola之重要因子，且可應用於十字花科黑斑病之生物防治具優異之開發潛力。

The black leaf spot disease caused by Alternaria brassicicola has been one of the most troubled diseases on crucifers which occurred worldwide. The pathogen was known to affect the host plants on all growth stages including seeds for propagation. The main solution to reduce the pathogen infection has been mainly chemical fungicides that are noxious to the environment and consumers as well. Development of bio-fungicides as an alternative has been attempted because the great concern on the chemical residue on the freshly consumed vegetables. The main of my objectives were to identify and characterize an antifungal compound produced by Taiwan native antagonistic Bacillus subtilis TKS1-1 for the control of the black spot disease on Crucifers, and the mode of action. In this investigation, 4 BSG strains shown with superior antagonistic activity against A. brassicicola in a preliminary dual culture assay namely TKS1-1, SP4-17, WG6-14 and WP8-12 were screened for effectiveness as the microbial agent for controlling black spot disease on crucifers. The Bacillus subtilis TKS1-1 culture in PSB for 5 days culture filtrate was among the test strains the best for antagonistic activity. The application of TKS 1-1 culture filtrate at different periods of spraying on controlling disease of detached cabbage leaf infection A. brassicicola, the pathogen was conducted one day after inoculation with the culture filtrate has loss control to infection, otherwise at the same time and one day before inoculation with the culture filtrate did not cause any symptoms on the cabbage leaf surface. The culture filtrate was very stable to heat and insensitive to pH. They still retained strong antifungal activity after treatment at pH values ranging from 2 to 13 for 24 hrs or at 100 ℃ for 20 min. The culture filtrate inhibitory effect was manifested by the delayed spore germination, the significantly deterred and extensively malformed germ tube development by in vitro system, as well as in vivo system. It’s worth noting that the germ tube malformation was apparently associated with the incapability of appressorium-like structure formation. The culture filtrate effect on A. brassicicola mycelia quickly loses their regulatory control on the membrane permeability indicating instant damage on the membrane cause to electrolyte leakage was increased 2.1 folds at 4 hours post treatment and reached maximum increase of 3.1 folds at 28 hours post treatment. To identify and characterize an antifungal compound produced byTKS1-1, extraction of the antibiotics was attempted by different (n-butanol, ethyl acetate, Sep-pakR C18, acid precipitation) extraction methods. The n-butanol extraction method was the most effective method, by thin layer chromatography (TLC) showed the antifungal activity were detected at Rf values of 0.06, 0.15, 0.36, and the color reaction indicated their amino acid and lipid containing characteristics. Another extraction method was purified by Sep-pak C18R cartridge clarification with the use of 70% methanol as eluent. Further purification of the antibiotic by reverse phase high performance liquid chromatography (HPLC) separation of retention times were 3.43 min and 3.98 min with nearly iturin A standard identical UV absorption spectrum. The molecular characterization of the HPLC purified antibiotics by electrospray ionization quadruple (Q-trap) tandem mass spectrometry analysis by whole span scanning revealed the detection of 3.43 min the major ions at 1043 m/z, and b-type ions b2 (212 m/z), b3 (299 m/z), b4 (524 m/z), b5 (638 m/z), b6 (801 m/z), b7 (915 m/z), and proven to be iturin A2 (C14); 3.98 min the major ions at 1057 m/z, and b-type ions b2 (212 m/z), b3 (299 m/z), b4 (538 m/z), b5 (652 m/z), b6 (815 m/z), b7 (929 m/z), and proven to be iturin A3,4,5 (C15). In the pest reports, the fatty acid length of iturins is known to be important for their antifungal activity which increases with increasing number of carbon atom, in my experiments with the same results. The above results indicate that antibiotic iturin A produced by TKS1-1 played an important role in antagonism against A. brassicicola. Therefore, B. subtilis TKS1-1 could be a useful organism for development of a bio-fungicide agent for bio-control of leaf spot on crucifers. 

目錄

中文摘要-------------------------------------------------------------------------------------------Ⅰ
英文摘要-------------------------------------------------------------------------------------------Ⅲ
目錄-------------------------------------------------------------------------------------------------Ⅵ
表次目----------------------------------------------------------------------------------------------Ⅸ
圖次目----------------------------------------------------------------------------------------------Ⅹ
壹、前言----------------------------------------------------------------------------------------------1
一、十字花科蔬菜之經濟重要性-------------------------------------------------------------1
二、十字花科黑斑病菌之發生與危害-------------------------------------------------------1
三、十字花科黑斑病菌之研究概況----------------------------------------------------------2
四、 十字花科黑斑病之防治應用------------------------------------------------------------3
五、 生物防治(Biological control)------------------------------------------------------------4
六、芽孢桿菌屬 (Bacillus spp.) 之特性與應用概況-------------------------------------4
(一) Iturins family之特性與防治應用----------------------------------------------------6
(二) Surfactin family之特性與防治應用------------------------------------------------7
貳、 材料與方法----------------------------------------------------------------------------------10
一、 供試藥品來源----------------------------------------------------------------------------10
二、 病原菌來源、保存方式------------------------------------------------------------------10
三、 培養液與培養基之製備----------------------------------------------------------------10
四、 具優異拮抗性枯草桿菌群Bacillus subtilis group菌株之篩選------------------11
五、 不同培養基對TKS1-1拮抗活性表現之影響---------------------------------------12
六、 抗生物質對黑斑病菌侵入感染之影響----------------------------------------------12
七、 溫度對拮抗物質活性之影響----------------------------------------------------------13
八、 酸鹼度對拮抗物質活性之影響-------------------------------------------------------13
九、 拮抗物質對Alternaria brassicicola孢子發芽影響--------------------------------13
十、 枯草桿菌 TKS1-1培養濾液對黑斑病菌孢子細胞膜通透性之影響----------14
十一、 枯草桿菌TKS1-1抗生物質之萃取及對黑斑病菌之拮抗活性測試--------15
(一) 逆相式層析淨化法(Sep-pakR C18)-----------------------------------------------15
(二) 酸沉降萃取法------------------------------------------------------------------------15
(三) 有機溶媒萃取法---------------------------------------------------------------------16
十二、抗生物質之薄層色層分析 (Thin Layer Chromatography，TLC) 分離及生物活性檢測-------------------------------------------------------------------------------16
十三、高效能液相層析儀 (High Pressure Liquid Chromatography，HPLC) 分析iturin A ------------------------------------------------------------------------------------17
十四、串聯式質譜儀 (MS/MS) 分析iturin A--------------------------------------------17
參、 結果-------------------------------------------------------------------------------------------19
一、 具優異拮抗性枯草桿菌菌株之篩選-------------------------------------------------19
二、 不同培養液對供試菌抗生活性表現之影響----------------------------------------19
三、 拮抗菌處理時間對病原菌侵入感染甘藍葉片之影響----------------------------19
四、 溫度對抗生物質活性之影響----------------------------------------------------------20
五、 酸鹼值對抗生物質活性之影響-------------------------------------------------------20
六、 TKS 1-1培養濾液對黑斑病菌孢子發芽影響--------------------------------------21
七、 TKS1-1培養濾液對黑斑病菌細胞膜功能的影響---------------------------------21
八、 經Sep-PakR C18淨化處理沖提液所含抗生物質對病原菌菌絲、孢子發芽及感染能力之影響-------------------------------------------------------------------------22
九、 不同萃取方式之抗生物質萃取液對黑斑病菌菌絲、孢子發芽及感染能力之影響----------------------------------------------------------------------------------------23
十、 於寄主植物施用抗生物質對黑斑病菌侵入感染寄主作用影響----------------24
十一、 n-Butanol萃取液之薄層色層分析法---------------------------------------------24
十二、 Iturin A 鑑定--------------------------------------------------------------------------25
肆、 討論-------------------------------------------------------------------------------------------27
伍、參考文獻--------------------------------------------------------------------------------------35
陸、圖表說明--------------------------------------------------------------------------------------50
柒、附錄--------------------------------------------------------------------------------------------74

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