Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89333
標題: 植物內生細菌Bacillus amyloliquefaciens SPX1於番茄組織內的纏據及防治番茄青枯病的效果
Colonization of bacterial endophyte-Bacillus amyloliquefaciens SPX1 in tomato and its potential for controlling bacterial wilt
作者: Hui-Ru Pan
潘蕙如
關鍵字: 內生細菌
Bacillus amyloliquefacines SPX1
番茄
纏據
青枯病
endophytic bacteria
Bacillus amyloliquefacines SPX1
tomato
colonization
bacterial wilt
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摘要: 內生細菌為能纏據於植物組織內,且不會對寄主造成任何病徵的細菌。本研究供試之內生細菌為,來自葉用甘藷組織的液化澱粉芽孢桿菌Bacillus amyloliquefacines SPX1菌株。比較不同浸漬時間,以胚軸剪斷法將SPX1菌株導入番茄組織,得知剪胚軸番茄於浸漬時間超過30 min後,於0-6 cm組織部位所導入的SPX1族群量無顯著差異,且導入之菌量與穩定性較土壤澆灌法佳。培養特性測試結果顯示,SPX1菌株於添加蔗糖、果糖、穀氨酸、氯化銨及硫酸銨之察氏培養基 (Czapek's medium) 中生長良好,且在低於8 %含鹽 (NaCl) 量與pH 介於5-9的LB培養基 (Lysogeny broth) 中生長良好,即SPX1能於一般的栽培環境或pH值較低的環境條件下生存。於寄主範圍測試,顯示以澆灌法將108 cfu/ml之SPX1懸浮液澆灌於葉用甘藷、番茄、豇豆及胡瓜等作物根圈,接種14天之後,皆可於所有作物莖基部組織測到該菌株,分離率介於80至100 %。趨化性的測試顯示,蔗糖、果糖、精胺酸及酪胺酸能明顯使SPX1產生趨化性。評估添加SPX1菌株防治番茄青枯病,顯示以胚軸剪斷法導入SPX1菌株之番茄幼苗,於含106 cfu/g soil濃度青枯病菌感染土28天後,得知接種SPX1菌株之番茄罹病度可由95 %降至62.5 %,與對照組有顯著差異 (p≦0.05)。然以土壤澆灌法施用SPX1菌株懸浮液之番茄幼苗,則無法於含高濃度青枯病菌土壤中展現防治效果。本研究以胚軸剪斷法配合澆灌法施用SPX1菌株,可提升防治青枯病效果,罹病度可由96.4 %降低至46.4 %,與對照組有顯著差異 (p≦0.05)。另分別以胚軸剪斷法與土壤澆灌法,將SPX1菌株導入聖女與金英番茄苗,結果顯示,添加SPX1菌株亦能降低兩種番茄之青枯病罹病度。此外,以胚軸剪斷法將SPX1菌株分別導入葉用甘藷與番茄苗,指出接種28天後,於葉用甘藷地上部12 cm處的莖部組織可測得該菌株,以0-4 cm組織部位之菌量最高約104 cfu/g tissue;於番茄苗導入SPX1菌株的處理,顯示該菌株亦能分佈於全株番茄之莖部組織,菌量集中於0-3 cm處,族群量約為103 cfu/g tissue。比較不同品種番茄導入SPX1菌株之菌量變化,得知SPX1菌株於聖女與金英番茄組織內之菌量較農友301高,且於0-3 cm組織部位的族群量達105 cfu/g tissue。為觀察SPX1在番茄組織中分布的位置,利用轉型嵌入綠螢光基因而得到的SPX1/DH5α (pAD43-25) 轉型菌株,澆灌於農友301番茄苗,顯示轉型菌株可纏據於番茄根部組織的表皮細胞,且於側根的分叉點有大量的菌體存在。持續以胚軸剪斷法將轉型菌株強勢導入番茄組織,7天後利用螢光顯微鏡觀察,得知轉型菌株主要分布於木質部,可經由細胞間隙向皮層、表皮轉移並生長。由上述結果得知,來自於葉用甘藷之內生細菌B. amyloliquefacines SPX1菌株,具有降低番茄青枯病之效果,該菌株可由番茄根系進入植株,並透過細胞間隙移行;此外,SPX1菌株具有內寄生於他種作物之能力,未來可於溫室或田間進一步評估防治不同作物萎凋性病害之潛力。
Endophytic bacteria (EB) invades the living plants tissues without causing apparent symptoms of diseases; meanwhile, several EBs can protect host from phytopathogens. The EB of Bacillus amyloliquefacines SPX1 isolated from sweet potato was used in this study. The results of introduction tests indicated that the populations and colonization area of SPX1 strain had no significant difference at 0-6 cm tissues site of tomato after inoculated into plant tissues by hypocotyl cutting inoculation method for 30 or 120 min. Besides, hypocotyl cutting inoculation method was better than drenching method for SPX1 strain colonizing in tomato tissues. Nutrient utilization showed that SPX1 preferred in the media added with carbon source such as sucrose and fructose, and nitrogen sources such as glutamate, ammonium chloride and ammonium sulfate. Moreover, SPX1 also can grow in lysogeny broth cantained NaCl ranged from 0 to 8 %. The pH test showed that pH 5 to 9 were suitable for the growth of SPX1. Host range tests pointed out that the reisolation rate of SPX1 strain could reach 80 to 100 % in sweet potato, cucumber, tomato, asparagine bean, pepper, and melon at 14-day after inoculation. Chemotaxis assays revealed that SPX1 is specifically attracted by sucrose, fructose, arginine and tyrosine. In greenhouse experiment, the SPX1 strain only showed the ability in controlling the bacterial wilt (BW) of tomato with hypocotyl cutting inoculation method, and could decrease the disease severity by 32 % at 28-day after transplated into the Ralstonia solanacearum-infested soil (106 cfu/g soil). Furthermore, the SPX1 strain treatments could reduce the disease severity of BW by 50 % by using hypocotyl cutting inoculation combined with drenching inoculation method. The ability effect of SPX1 in controlling BW also shown the same efficacy in different tomato cultivars (Santa and Hualien Asveg 21) with drenching and hypocotyl cutting inoculation method. The experiment of distribution efficacy of SPX1 strain showed that SPX1 could colonize in the whole stem tissues of vegetable sweet potato and tomato at 28-day after inoculation; however, the population of SPX1 could reached 103-104 cfu/g tissue at the basal parts of the inoculation site (0-3 or 4 cm) than in higher tissues site based on hypocotyl-cutting seedlings. Morever, the colonization ability of SPX1 on different tomato cultivar showed that the SPX1 more preferred colonized in Santa and Hualien Asveg 21 than Farmers 301. In order to observing the distribution of SPX1 in tomato plant tissues, the transformed line, SPX1/DH5α (pAD43-25) with green fluorescent protein marker, was used in this study. The SPX1/DH5α (pAD43-25) could be observed on root surface and colonize on axils of lateral roots dominantly. Furthermore, the SPX1/DH5α (pAD43-25) basically colonized in xylem tissues, and transmitted intercellarly from xylem to cortex, and trichome of epidermis at 7-day after inoculation with hypocotyl cutting inoculation method. Thus, the B. amyloliquefaciens SPX1 strain could be used as a biocontrol agent in controlling BW of tomato in greenhouse experiment and colonize in tomato tissue stably. On the other hand, the SPX1 could colonized in several crops, and the controlling ability of BW on each crop should be further studied.
URI: http://hdl.handle.net/11455/89333
文章公開時間: 2017-08-28
Appears in Collections:植物病理學系

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