臺灣博碩士論文加值系統

萵苣猝倒病是萵苣苗期的重要病害之一，它的主要病原菌是 Pythium aphanidermatum Edson，可經由栽培介質傳播，因此如何確保栽培介質免於病原菌污染，是防治本病害的一種重要手段。本試驗主要目的在於探討產氣細菌用於防治萵苣猝倒病的可行性。從台灣中部農田與植株根圈土分離產氣菌株，比較它們產氣抑制 P. aphanidermatum PA01 菌絲生長之效果後，選取抑菌較強之 E006、E010 及 P013 菌株進行生理生化測試與 Biolog 鑑定系統測試，結果將 E006 及 E010 鑑定為 Enterobacter 屬；P013 鑑定為 Pseudomonas putida。利用培養皿盤對扣檢測法測試 King’s medium B、Nutrient agar、Yeast malt extract agar、Luria-Bertani medium 及 Potato dextrose agar 等五種培養基對 E006、E010 及 P013 等三菌株產生氣體抑制本病菌的效果，結果顯示各菌株生長在 KB 培養基上，均具有較佳的抑菌效果。進一步修正 KB 培養基配方，以測試其各組成分對產氣抑菌效果的影響，結果發現配方中的 proteose peptone No.3 是產氣菌產生抑菌效果的關鍵成分。嘗試利用 14 種土壤添加物替代 KB 培養基中的 proteose peptone No.3，發現 2 % 苜蓿種子粉、苜蓿乾草粉與肉骨粉能使產氣菌株產生抑制 P. aphanidermatum PA01 菌絲生長的氣體。進一步，以氣相層析質譜儀分析三菌株分別於 KB 培養基、苜蓿種子粉培養基、苜蓿乾草粉培養基或肉骨粉培養基中生長所產生氣體的種類，結果計有： styrene、1-Undecene、benzoic acid, 2-hydroxy-, methyl ester、methane, thiobis-、cyclopropane, 1-methyl-2-pentyl-、benzyl methyl sulfide、2-phenyl-2,4-octadienol 及 phenol,2,6-bis(1,1-dimethylethyl)-4-methyl- 等，其中 benzoic acid, 2-hydroxy-, methyl ester、methane, thiobis- 與 benzyl methyl sulfide 均具有抑菌的效果。在含有病原菌的介質中單獨添加 0.5 % (w/w) 苜蓿種子粉、苜蓿乾草粉及肉骨粉等添加物，或僅接種產氣細菌等處理，皆無法有效防治萵苣猝倒病；然而若將苜蓿種子粉或苜蓿乾草粉與產氣細菌 Enterobacter sp. E010 一併均勻拌入含有病原菌的介質中，然後密封燻蒸處理6 天後，再播種萵苣種子，則可達到 90 % 左右的防治率。

Damping-off caused by Pythium aphanidermatum Edson is a serious disease of lettuce seedlings grown in the culture medium in central Taiwan. The culture medium could be countaminated with the pathogen. Thus, preventing culture medium from contamination of the pathogen should play an important method in controlling the disease. Five gas-producing bacterial strains were isolated from field and rhizophere soils. Three strains E006, E010 and P013 were more effective in inhibiting mycelial growth of P. aphanidermatum PA01. Strains E006 and E010 were identified as Enterobacter spp.. Strain P013 was identified as Pseudomonas putida. Effective evaluations of gas-producing bacteria on plates of King’s medium B, Nutrient agar, Yeast malt extract agar, Luria-Bertani medium and Potato dextrose agar for suppression of mycelial growth of P. aphanidermatum PA01 were tested. Three strains grown on King’s medium B expressed better effectiveness in inhibiting the pathogen. Proteose peptone No.3 was the major ingredient in King’s medium B for the bacterial producing volatile compounds to inhibit the pathogen. Alfalfa seed meal (ASM), alfalfa hay meal (AHM) and bone meat meal (BMM) were respectively used to replaced proteose peptone No.3 in King’s medium B. Then, modified media were made and cultured E006, E010 and P013 for studying their suppressive ability to the pathogen. Volatile compounds released from gas-producing bacteria grown onto the King’s medium B, ASM medium, AHM medium and BMM medium were analyzed by Gas Chromatography. Volatile compounds were identified as styrene, 1-Undecene, benzoic acid, 2-hydroxy-, methyl ester, methane, thiobis-, cyclopropane, 1-methyl-2-pentyl-, benzyl methyl sulfide, 2-phenyl-2,4-octadienol and phenol,2,6-bis(1,1-dimethylethyl)-4-methyl-. Benzoic acid, 2-hydroxy-, methyl ester, methane, thiobis- and benzyl methyl sulfide were effective in inhibiting the pathogen. The culture media contaminated with the pathogen were treated with Enterobacter spp. E006, E010 and Pseudomonas putida P013 and/or organic amendments in the PE plastic bag. Enterobacter sp. E010 mixed with ASM or AHM was significantly effective in reducing 90 % disease incidence of lettuce damping-off caused by P. aphanidermatum in the greenhouse.

目錄
摘要……………………………………………………………….…....….i
Abstract………………………………………………………….…….….iii
目錄………………………………………………………….………...…..v
表目錄………………………………………………………….………...ix
圖目錄………………….………………………………………………....x
壹、前言…………………………………………………………….....…1
貳、材料與方法…………………………………………………………..7
一、供試植物來源…………………………………………………..7
二、病原菌分離及病原性測定……………………………………..7
(一)、病原菌的分離…………………………………………7
(二)、病原性測定……………………………………………8
三、病原菌鑑定…………………………………………………….8
四、游走孢子接種源的製備……………………………………….9
五、接種源濃度對萵苣幼苗猝倒病發生的影響...…………….….9
六、產氣菌株的來源與篩選………………………………………..9
七、產氣細菌菌株的鑑定…………………………………………11
(一)、生理生化測試………………………………………..11
(二)、Biolog 細菌鑑定系統………………………………..11
(三)、16S rDNA 核苷酸序列分析…………………………12
1. 16S rDNA序列之增幅……………………………….12
2. DNA片段回收及純化………………………………..13
3. 16S rDNA 核苷酸序列分析……………………..…..14
八、不同培養基質對產氣細菌抑菌效果的影響…………………14
九、修正培養基配方對產氣菌株抑菌效果的影響………………15
(一)、修正 KB 培養基配方的影響……………………….15
(二)、有機添加物對產氣細菌抑制病原菌生長的影響…..15
十、不同有機添加物培養產氣細菌對於
Pythium aphanidermatum PA01 產生游走子的影響.…..…16
十一、Enterobacter spp. E006、E010 及
Pseudomonas putida P013 菌株產生氣體之種類分析….17
十二、氣體標準品對於 P. aphanidermatum PA01 菌絲生長
的影響………………………………………………….....18
十三、萵苣猝倒病的防治試驗…………………………………..19
(一)、不同有機添加物防治萵苣猝倒病的效果
評估……………...…………………………………19
1.有機添加物對萵苣鮮重及株高的影響………………19
2.不同有機添加物的防治效果…………………………19
(二)、產氣細菌防治萵苣猝倒病之試驗…………………..20
1. 產氣細菌對萵苣生育的影響……………………..…20
(1) 產氣細菌對萵苣種子發芽的影響…………..……20
(2) 產氣細菌對萵苣生育的影響…………...………...20
2.產氣細菌的防治效果…………………………………21
(三)、產氣細菌配合有機添加物防治萵苣猝倒病
之試驗…………………………………………...…22
叁、結果………………………………………………………………..23
ㄧ、病原菌鑑定………………………………………………..…23
二、接種源濃度與萵苣幼苗猝倒病發生率間的關係..…………23
三、比較產氣細菌菌株之抑菌效果…………………..…………24
四、菌株鑑定………………………………………………..……24
(一)、生理生化測試…………………………………….…24
(二)、Biolog 細菌鑑定系統………………………………25
(三)、16S rDNA 核苷酸序列分析………………………..25
五、不同培養基質對 Enterobacter spp. E006、E010 及
Pseudomonas putida P013 產氣抑菌效果的影響..…..….25
六、修正培養基配方對產氣細菌抑菌效果的影響……………..26
(一)、修正 KB 培養基配方對產氣細菌菌株抑菌
效果的影響………………………………...………26
(二)、有機添加物對產氣菌株抑菌效果的影響……….…27
七、有機添加物配合產氣細菌對於 P. aphanidermatum PA01
產生游走子的影響………………….……………………...27
八、Enterobacter spp. E006、E010 及 Pseudomonas putida P013 菌株產生之氣體種類分析…………………….…………...28
九、氣體標準品對於 P. aphanidermatum PA01 菌絲生長的
影響…………………………………………………………28
十、不同有機添加物防治萵苣猝倒病的效果…………..………29
(一)、不同有機添加物對萵苣生育的影響……….………29
(二)、不同有機添加物的防病效果………………….……29
十一、產氣細菌防治萵苣猝倒病的效果………………………..30
(一)、產氣細菌對萵苣生育的影響…………………….…30
1. 產氣細菌對萵苣種子發芽的影響……………….….30
2. 產氣細菌對萵苣植株生育的影響…………………..30
(二)、產氣細菌的防病效果……………………….…...…..30
(三)、產氣細菌配合有機添加物防治萵苣猝倒病之
效果………………………..…………………….…31
肆、討論………………………………………………………………...32
伍、參考文獻……………………………………………………….…..39


























表目錄
表一、各產氣細菌菌株對萵苣猝倒病菌菌絲生長的影響……..………47
表二、細菌 E006、E010 及 P013 菌株之生理生化特性……………...48
表三、不同培養基質培養Enterobacter spp. E006、E010 及 Paeudomonas putida P013 等菌株所產生之揮發物對萵苣猝倒病菌菌絲生長的影響…………………………………………...49
表四、比較 King’s B 培養基配方組成份對 Enterobacter spp. E006、E010 及 Pseudomonas putida P013 菌株產氣抑制 Pythium aphanidermatum PA01 菌絲生長效果的影響………………....50
表五、有機添加物配合 Enterobacter spp. E006、E010 及 Pseudomonas putida P013 菌株產氣影響 Pythium aphanidermatum PA01 產生游走子之效果…………………………………………...........51
表六、產氣細菌 Enterobacter spp. E006、E010 及 Pseudomonas putida P013 菌株在不同培養基上分別產生氣體的種類數………….52
表七、氣相層析質譜儀分析 Enterobacter spp. E006、E010 及 Pseudomonas putida P013 菌株生長在不同培養基上所產生之主要氣體………………………………………………………...53
表八、Enterobacter spp. E006、E010 及 Pseudomonas putida P013 菌株生長在不同培養基上所產生的8種主要氣體及其波鋒面積的百分比大小………………………………………………….......54

圖目錄
圖一、萵苣幼苗猝倒病菌 (Pythium aphanidermatum PA01 菌株)
之接種源濃度與植株發病百分率間的關係………………….55
圖二、不同有機添加物培養基影響 Enterobacter sp. E006
菌株產氣抑制Pythium aphanidermatum PA01 菌絲生長的效 果……………………………………………………………....56
圖三、不同有機添加物培養基影響 Enterobacter sp. E010
菌株產氣抑制Pythium aphanidermatum PA01 菌絲生長的效果……………………………………………………………….57
圖四、不同有機添加物培養基影響 Pseudomonas putida P013
菌株產氣抑制Pythium aphanidermatum PA01 菌絲生長的效果…………………………………………………………….....58
圖五、苜蓿種子粉培養基上生長之 Enterobacter spp. E006、
E010 及 Pseudomonas putida P013 菌株產生之氣體的氣相層析質譜圖…………………………………………………….....59
圖六、苜蓿乾草粉培養基上生長之 Enterobacter spp. E006、
E010 及 Pseudomonas putida P013 菌株產生之氣體的
氣相層析質譜圖…………………………………………….....61
圖七、肉骨粉培養基上生長之 Enterobacter spp. E006、
E010 及 Pseudomonas putida P013 菌株產生之氣體的
氣相層析質譜圖…………………………………………….…63
圖八、King’s B 培養基上生長之 Enterobacter spp. E006、
E010 及 Pseudomonas putida P013 菌株產生之氣體的
氣相層析質譜圖………………………………………………..65
圖九、不同氣體標準品對 Pythium aphanidermatum PA01
菌絲生長的抑制效果………………………………………....67
圖十、在溫室中以不同有機添加物處理土壤後對萵苣植株
鮮重與株高之影響……………………………………………68
圖十一、不同有機添加物防治萵苣猝倒病之效果………………….69
圖十二、在溫室中以 Enterobacter spp. E006、E010 及 Pseudomonas putida P013 菌株之水懸浮液澆灌土壤後對萵苣植株鮮重與株高之影響…………………………………………………..70
圖十三、Enterobacter spp. E006、E010 及 Pseudomonas putida P013 菌株防治萵苣猝倒病之效果…………………………………..71
圖十四、Enterobacter spp. E006、E010 及 Pseudomonas putida P013 菌株配合不同有機添加物防治萵苣猝倒病之效果………...72
圖十五、Enterobacter spp. E010 菌株配合 0.5 % 苜蓿種子粉或苜蓿乾草粉防治萵苣猝倒病之效果。…………………………...74

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