Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/96242
標題: Isolation and characterization of bacteria associated with medicinal plant Platycodon grandiflorus roots
藥用植物桔梗根圈與根內細菌之分離及特性研究
作者: Cong-Min Huang
黃聰閔
關鍵字: 論文
桔梗
根內菌
內生細菌
Thesis
Platycodon grandiflorus
endophytes
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摘要: 農業上化學肥料以及農藥之使用雖然有效地提高了作物之產量,但長期並過量施用造成地力退化以及對生態造成影響,且所使用之資源為不可再生性,終有消耗殆盡之日。隨著環保及永續議題日益受到重視,永續農耕成為了一個熱門之研究方向,故微生物肥料和農藥之開發,可藉由減少化學肥料和農藥之施用,並提高作物之產量而達到永續及環保之目的。本研究旨在挑選由中草藥植物桔梗,其組織內部之微生物,並測試其對於植物生長促進之潛力,以及推估在植體中之活動和與宿主之互動關係,並於種植試驗中回接以及回篩,測試是否在實際上能對植物之生長有幫助以及助益能力之多寡。 從桔梗之根圈以及根部中,共挑選出70株菌,包含45株根圈以及25株根內菌,根圈菌以Bacillis、Burkholderia、Microbacterium、Pseudomonas和Rhizobium為主;根內菌則是以Burkholderia、Pantoea和Rhizobium篩選出之數量為多。在促進植物生長 (PGP) 測試中,溶磷活性測試之20株菌中有3株測試菌株表現較高之活性,高於300 μg/ ml之上;17株測試IAA產量之菌株中,7株菌之活性高於50 μg/ ml ;測試之16株菌中有5株測試菌株具較高之固氮活性,於1.20至2.34 nmol/ (tube ず hr)之間。由上述PGP測試、親緣分析並比對菌株之生物安全分級後,從中挑選6株菌進行後續測試,分別為Microbacterium sp. NAI1、Rhizobium sp. NAI3、NAI4、NAI5和NFI1以及Xenophilus sp. NFI5。 桔梗種子生物分析中,相對於未接菌之對照組,NFI1可幫助胚莖生長,而NFI5可促進胚根發育,其餘接種菌株之處理無顯著差異;基質水解測試中NAI1可以水解果膠、纖維素以及澱粉,且活性強,NAI3與NFI1可水解果膠及纖維素,NFI5可水解果膠,NAI4和NAI5可水解纖維素;碳源利用分析中,NAI1偏好使用碳源數量多,且對於養分之利用能力強,NAI3、NAI4、NAI5和NFI1僅偏好利用碳水化合物類型碳源, NFI5則偏好利用羧酸和聚合物類型碳源;在0.3、0.5和1% 洋菜濃度之培養基上移動性試驗結果,僅NAI3、NFI1和NFI5為正反應,由上述實驗再從中挑選NAI1、NAI4、NFI1和NFI5進行桔梗之蛭石瓶和盆栽試驗。 蛭石三角瓶介質栽培50天之試驗中,接種NAI4和NFI5之處理相對於對照組而言,植物根部之鮮重較佳但統計上無顯著差異,並由回篩中所接種之菌株皆有進入組織內部。盆栽試驗中可證明NFI1有進入植物組織內部,接種NAI4與添加半量化肥之處理相對於半量化肥組,地上部生質量、總多酚含量和抗氧化活性較高,而試驗之所有植株皆無表現病徵,但結果中相對於半量化肥組,接種NAI1並添加半量化肥之處理根部生長較差、接種NFI1並添加半量化肥之處理地上部生長較差,推估與同時具碳水化合物水解酵素活性以及在植物組織內之生長特性有關。故除微生物所表現之PGP特性外,推測菌株之生長特性也會影響植物在生長中之表現。
Chemical fertilizers and pesticides are commonly used to maintain soil fertility, reduce plant pathogen and pest. But long-term and overdose usages of agrochemicals have lead to soil degradation and pollution. To achieve sustainable agriculture, biofertilizers and biopesticides which are environment friendly provide alternatives to promote plant growth directly or indirectly. The present study was undertaken to explore root associated bacteria from medicinal plant Platycodon grandiflorus. Samples were collected from rhizosphere and root interior and plated on nutrient agar, nitrogen-free agar or tricalcium phosphate agar. After isolation all isolates were subjected to 16S rDNA sequencing and phylogenetic analysis. Plant growth promoting traits regarding nitrogen fixation, phosphate solubilization or IAA production were determined. Besides, the plant constituent decomposing and carbon source utilizing abilities of selected isolates were also demonstrated. The superior isolates were used in seed bioassay and pot experiment to evaluate their performance on plant growth. Among 45 isolates obtained from rhizosphere, the dominant bacterial genera consist of Bacillus, Burkholderia, Microbacterium, Pseudomonas and Rhizobium. A total 25 isolates mainly affiliated with genera Burkholderia, Pantoea and Rhizobium were obtained from root interior. Some of these isolates showed higher phosphate solubilizing activity (> 300 μg ml-1) or IAA producing activity (> 50 μg ml-1). Considered for their plant growth promoting traits and biosafety level, six isolates namely Microbacterium sp. NAI1, Rhizobium sp. NAI3, NAI4, NAI5, NFI1 and Xenophilus sp. NFI5 were selected and further characterized. Seed bioassay demonstrated that strain NFI1 increase growth of epicotyl while strain NFI5 promote growth of radicle after 10 days of cultivation. Strain NAI1 showed prominent utilization of cellulose, pectin, starch and xylan, and a variety of carbohydrates were well utilized. As for Rhizobium spp., only cellulose was utilized by 4 isolates and no remarkable carbon utilization were revealed within 48 h of cultivation. Strain NFI5 showed pectin decomposing activity and several organic acids were weak utilized. In vermiculite containing flask, inoculation of strain NAI4 and NFI5 increased fresh weight of Platycodon grandiflorus after 50 days of cultivation. All these inoculants were obtained and confirmed by BOX-PCR fingerprinting after re-isolating from root interior. In pot experiment, treatment NPK + strain NAI4 or NPK + strain NFI5 increased fresh weight and dry weight of stems and leaves. The polyphenols content and antioxidative activities of Platycodon grandiflorus roots were the highest in treatment NPK + strain NAI4 after 4 months of cultivation. However, inoculation of strain NAI1 decreased biomass of roots while strain NFI1 decreased biomass of stems and leaves. From the present study it is proposed that carbon utilizing traits of bacteria should also be taken into considerations when developing superior bioinoculants.
URI: http://hdl.handle.net/11455/96242
文章公開時間: 10000-01-01
Appears in Collections:土壤環境科學系

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