TWI818611B - 地衣芽孢桿菌ec34-01生物膜形成組成物及其應用 - Google Patents
地衣芽孢桿菌ec34-01生物膜形成組成物及其應用 Download PDFInfo
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Abstract
本發明係關於一種包含地衣芽孢桿菌菌株EC34-01(Bacillus licheniformis EC34-01)培養物之生物膜形成組成物,特徵在於該培養物係藉由將地衣芽孢桿菌EC34-01菌株以天然資材組成之液態培養基進行培養而成。本發明之地衣芽孢桿菌EC34-01組成物可於不結球白菜葉表及根系纏據形成生物膜,藉以提升不結球白菜對乾旱與淹水逆境之耐受性,並且具有降解化學農藥,例如馬拉松、第滅寧等的功效。
Description
本發明係關於一種具有生物膜形成能力的組成物,更特別地係關於一種地衣芽孢桿菌EC34-01組成物,包含具優異生物膜形成能力之地衣芽孢桿菌EC34-01(Bacillus licheniformis EC34-01)菌株,經天然資材組成之液態培養基進行培養所得之培養物。本發明之地衣芽孢桿菌EC34-01組成物可用於提升不結球白菜對乾旱與淹水逆境之耐受性,並且具有降解化學農藥的功效。
生物膜(Biofilm)為微生物於環境中生存之自然形成現象,此生物膜可保護菌體免於受外部物理及化學因子之干擾,而具耐受環境壓力之能力,並有助其於植物體展現功能提升效用及適存性。已知有多種和植物相關之細菌(plant-associated bacteria)如,農桿菌(Agrobacterium tumefaciens)、枯草桿菌(Bacillus subtilis)、普氏假單孢菌(Pseudomonas putida)、螢光假單孢菌(Pseudomonas fluorescens)、玉米細菌性萎凋病菌(Pantoea stewartii)、苜蓿中華根瘤菌(Sinorhizobium melilotii)等,皆可在植物葉表、根圈或植物細胞與維管束內形成生物膜,此生物膜之形成亦被證實,在其是否能造成植物病害或保護植物、促進植物生長與抗病性等植物-微生物交互作用中扮演重要角色(Danhorn,T.,and Fuqua,C.Annu.Rev.Microbiol. 61:401-422,2007)。
Rudrappa等人研究發現,受丁香假單孢菌(Pseudomonas syringae)感染之阿拉伯芥根部會分泌蘋果酸(malic acid),誘引生物防治用根圈細菌B.subtilis FB17纏據於根上並形成生物膜(Rudrappa,T.,et al.Plant Physiol. 148:1547-1556,2008)。而本案發明人研究室亦證實,於接種潰瘍病原前一天噴灑枯草桿菌群TKS1-1及WG6-14菌株,可明顯抑制潰瘍病原於墨西哥萊姆葉表生物膜形成及纏據能力,且枯草桿菌之應用亦可明顯降低單位葉表面積柑桔潰瘍病斑數(Huang et al.,PLoS ONE 7:e42124,2012)。此等結果顯示,利用拮抗性枯草桿菌抑制潰瘍病原於葉表形成生物膜能力,可降低柑桔潰瘍病害發生。
於是,本發明利用由台灣環境中篩選分離得,具優異生物膜形成能力之地衣芽孢桿菌EC34-01菌株,將其以天然資材所組成之液態培養基進行培養,而得到一種地衣芽孢桿菌EC34-01組成物。並且,於本發明已證實,將此組成施用於不結球白菜,可於不結球白菜葉表及根系纏據形成生物膜,提升不結球白菜對乾旱與淹水逆境之耐受性。本發明亦進一步確定,本發明之地衣芽孢桿菌EC34-01組成物不僅可用於保護植物健康、對抗逆境,並且具有分解化學農藥的能力,包括常用之農業殺蟲劑例如馬拉松、第滅寧等。
於一方面,本發明係關於一種於不結球白菜形成生物膜之地衣芽孢桿菌EC34-01組成物,其包含一種將地衣芽孢桿菌EC34-01(Bacillus licheniformis EC34-01)於一包含1.0-2.0%黃豆粉、0.5-1.0%酵母粉、及0.5-2.0%黑糖等天然資材,以重量計(% W/L),之液態培養基中於30℃下培養5-10天所製成之培養物。
於本發明之一具體實施例,所述之液態培養基係包含1.0%黃豆粉、0.5%酵母粉、2.0%黑糖,以重量計(% W/L)。
於本發明之一些具體實施例,所述之生物膜形成組成物係用於在不結球白菜葉表及根系纏據形成生物膜。
於本發明之一些具體實施例,所述之組成物係用於提升不結球白菜之耐乾旱能力。
於本發明之一些具體實施例,所述之組成物係用於降解農業副產物中殘留之化學農藥。
於本發明之一些具體實施例,所述之化學農藥係選自馬拉松及第滅寧。
圖1係顯示,枯草桿菌B.subtilis 168菌株、和地衣芽孢桿菌B.licheniformis EC34-01菌株於24孔聚苯乙烯盤的孔中形成生物膜能力之評估結果。
圖2係顯示,地衣芽孢桿菌B.licheniformis EC34-01及枯草桿菌B.subtilis 168菌株於不結球白菜葉表上形成生物膜之能力。
圖3係顯示,施用枯草桿菌B.subtilis 168、地衣芽孢桿菌B.licheniformis EC34-01製劑對不結球白菜於6天乾旱後,植株生長之影響。以水及SYB培養液作為對照組。(A)植株鮮重;(B)植株乾重;(C)株高;(D)根長;(E)莖直徑。
圖4係顯示,地衣芽孢桿菌EC34-01製劑對於化學農藥馬拉松(A)及第滅寧(B)之降解能力,以農藥降解百分比(%效率)表示,隨著與菌株製劑的培養天數增加,農藥之降解比率增加。其中對照組(Ctrl)是未添加微生物菌株之製劑對照組。
本發明係提供一種包含地衣芽孢桿菌EC34-01(Bacillus licheniformis EC34-01)且具有生物膜形成能力之組成物。於本文所稱之”生物膜(biofilms)”係指,微生物附著於一物體表面所形成的薄狀構造,可保護微生物菌體免受外部物理及化學因子之干擾,且使之具環境壓力之耐受性。於本文所稱之”具有生物膜形成能力”係指,一微生物製劑施用於植物後,其中的菌體能夠群聚附著於植物體表面,如葉表或根圈而形成生物膜,提升該植物對抗諸如乾旱與淹水等逆境之耐受性。
本發明之其他特色及優點將於下列實施範例中被進一步舉例與說明,而該實施範例僅作為輔助說明,並非用於限制本發明之範圍。
實施例一、地衣芽孢桿菌EC34-01生物膜形成組成物之製備
將1毫升地衣芽孢桿菌EC34-01菌株(OD620=0.3),於25毫升胰蛋白酶大豆培養基(BDTM Trytic Soy Broth,TSB,Franklin Lakes,NJ,USA)中,30℃下以125rpm震盪培養8小時。之後取1毫升該TSB細菌培養物,加至100毫升SYB培養液(包含1.0-2.0%黃豆粉、0.5-1.0%酵母粉、及0.5-2.0%黑糖,以重量計(% W/L)中,於30℃下以125rpm震盪培養5天,而製備完成本發明之具有生物膜形成能力的地衣芽孢桿菌EC34-01組成物。
本發明之地衣芽孢桿菌EC34-01(寄存編號為:BCRC911132)是由中興大學植物病理學系植物病害管理研究室所提供,該菌株係分離自台灣雲林縣菱角田土壤,依16S rRNA序列分析、細菌生理生化特性測試、脂肪酸鑑定系統及Biolog分析,鑑定為地衣芽孢桿菌(Bacillus licheniformis),參見江嘉容(2011)檬果炭疽病的生物防治菌鑑定與其防病之雛形醱酵營養配方,國立中興大學植物病理學系碩士論文。
實施例二、地衣芽孢桿菌EC34-01生物膜形成組成物之植物保護功效評估
生物膜形成能力
將地衣芽孢桿菌B.licheniformis EC34-01及枯草桿菌B.subtilis 168菌株培養於Luria-Bertani broth(LB,BD,Franklin Lakes,NJ,USA)培養液中,於30℃下以150rpm震盪培養24小時,依微量多孔盤測定法(microplate assay)(Huang et al.J.Bacteriol. 188(8):3116-3120,2006)進行生物膜能力測試,每處理4重複。
結果顯示,地衣芽孢桿菌B.licheniformis EC34-01菌株於微量多孔盤測定中,其生物膜形成能力皆較已知生物膜形成能力缺失之枯草桿菌B.subtilis 168菌株為高(圖1)。
於不結球白菜葉表之生物膜形成能力
為評估本發明地衣芽孢桿菌EC34-01生物膜形成組成物於作物葉表生物膜形成且附著之能力,試驗中將各細菌製劑懸浮液(OD620=0.3)以噴瓶在不結球白菜之葉表及葉背各噴灑1ml細菌懸浮液,最後套袋保濕24小時。以直徑1cm打孔器取不結球白菜葉盤(三鳳二號品種不結球白菜,農友種苗股份有限公司,高雄市,台灣),利用0.025%吖啶橙溶液(0.025% acridine orange in 0.026 M citric acid buffer,pH 6.6)染色,以無菌水清洗後利用螢光顯微鏡進行影像拍攝,並以Image J軟體換算螢光區塊亮度,以作為菌體纏據能力之相對量值,每處理4重複。
結果顯示,地衣芽孢桿菌B.licheniformis EC34-01製劑於不結球白菜葉表之生物膜形成能力,明顯較枯草桿菌B.subtilis 168菌株為高(圖2)。
提升作物之耐淹水逆境能力
提升作物之耐乾旱逆境能力
將不結球白菜種子利用次氯酸鈉溶液表面消毒後,於無菌水中催
芽,其後將種子種植於裝有栽培介質(泥炭土:珍珠石=6:1)的三吋塑膠盆中,於播種後三天後,分別澆灌水、稀釋100倍之SYB培養液及本發明之地衣芽孢桿菌EC34-01組成物(菌量約3×109CFU/ml),施用2次後,進行6天的乾旱處理,其後將不結球白菜植株再澆灌水3天,根系經清洗後,測量植株生育性狀。本試驗每處理使用30株植株,作為重複。
由圖3之結果顯示,本發明之地衣芽孢桿菌EC34-01組成物可有效地在植株經乾旱逆境後,提升不結球白菜的植株存活率,且植株鮮重、乾重、株高、根長及莖徑,亦顯著高於水及SYB培養液對照組與生物膜形成能力較差之對照菌株B.subtilis 168處理組。此等結果表示,本發明之地衣芽孢桿菌(B.licheniformis)EC34-01生物膜組成物有助於提升不結球白菜的耐乾旱能力。
實施例三、地衣芽孢桿菌EC34-01生物膜形成組成物之農藥降解功效評估
台灣每年約有超過120萬公噸的農業副產物,包括糧作、蔬果等副產物,以及菇類栽培介質與畜牧養殖副產物等,雖已有許多將此類副產物製成堆肥再生循環利用的方案提出,但由於慣行的農業栽種與養殖方法,致使所產出的副產物可能有農藥殘留的風險。本實例遂進一步評估本發明之地衣芽孢桿菌EC34-01對於常用農藥的降解效率,以期可利用於分解再生循環資材中殘留的化學農藥。
將地衣芽孢桿菌EC34-01接種於Luria-Bertani(LB)培養基,置於30℃,250rpm震盪培養一天,將菌液調整成OD620=1,供農藥降解試驗使用。測試用農藥採市售產品進行,於MSgg液態培養基(5mM磷酸鉀(pH 7)、100mM Mops(pH 7)、2mM MgCl2、700μM CaCl2、50μM MnCl2、50μM FeCl3、1μM ZnCl2、2μM硫胺素、0.5%甘油、0.5%麩胺酸、50μg/ml色胺酸、50μg/ml苯丙胺酸)分別加入最終濃度為100ppm之50%馬拉松可濕性粉劑及2.4%第滅寧水懸劑,再
添加於上述LB培養之地衣芽孢桿菌EC34-01(最終菌液濃度為OD620=0.1),置於30℃、250rpm震盪培養,1、3、5日後,取出1mL進行農藥濃度分析,另以未添加地衣芽孢桿菌EC34-01之混合液為對照組(Ctrl)。馬拉松及第滅寧採用氣相層析串聯式質譜儀分析,將上述培養液分別以丙酮稀釋5000倍後分別進行分析,氣相層析串聯式質譜儀離子條件及層析條件如下:氣相層析儀串聯式質譜儀分析,質譜儀使用Bruker Scion TQ-436GC系統,層析管柱為Agilent HP-5MSi 30m×0.25mm,分析載氣為氦氣,以定壓模式進行,壓力設定為21psi,烘箱時間梯度設定初始溫度為70℃維持兩分鐘、其後以30℃/min升溫速率持續升溫至280℃、溫度到達後維持10分鐘,總分析時間為19分,進樣體積為3μl,注射口採不分流模式,溫度250℃。馬拉松(Malathion)檢測離子173→127及173→99,第滅寧(Deltamethrin)檢測離子253→93及253→174。
由圖4之結果顯示,地衣芽孢桿菌EC34-01組成物具有分解諸如馬拉松以及第滅寧等殺蟲化學農藥的能力,於第3天馬拉松可降解達77%、第5天則達100%完全降解;於第1天第滅寧即可降解53%以上,可用於再生循環資材,幫助分解農業副產物中殘留的化學農藥。
【生物材料寄存】地衣芽孢桿菌EC34-01(Bacillus licheniformis EC34-01)
寄存機構:財團法人食品工業發展研究所
寄存日期:111年6月6日
寄存號碼:BCRC911132
Claims (6)
- 一種於不結球白菜形成生物膜之組成物,包含地衣芽孢桿菌EC34-01(Bacillus licheniformis EC34-01)(寄存編號為:BCRC911132)於一包含1.0-2.0%黃豆粉、0.5-1.0%酵母粉及0.5-2.0%黑糖,以重量計(% W/L),之液態培養基中於30℃培養5-10天所成之培養物。
- 如第1項所述之組成物,其中該液態培養基係包含1.0%黃豆粉、0.5%酵母粉、2.0%黑糖,以重量計(% W/L)。
- 如第1項所述之組成物,其中該組成物係用於在不結球白菜葉表及根系纏據形成生物膜。
- 如第1項所述之組成物,其中該組成物係用於提升不結球白菜之耐乾旱能力。
- 如第1項所述之組成物,其中該組成物係用於降解農業副產物中殘留之化學農藥。
- 如第5項所述之組成物,其中該化學農藥係選自馬拉松、第滅寧及三賽唑。
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期刊 Jong-Hui Lim, Induction of Drought Stress Resistance by Multi-Functional PGPR Bacillus licheniformis K11 in Pepper, Plant Pathol J, 29(2) 2013 * |
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