請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/90147
標題: Diversity, physiological, biochemical and plant growth promoting characteristics of facultative oligotrophs
兼性少養分營細菌之多樣性、生理生化及植物生長促進特性研究
作者: Yi-Ying Kao
高薏盈
關鍵字: Facultative oligotrophs
兼性少養分營細菌
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摘要: Soil, a heterogeneous habitat harbors considerable quantities of oligotrophic bacteria which can proliferate with limited nutrients. But owing to the selective isolation of rapidly growing bacteria by conventional cultivation method, the exploration of oligotrophs seems limited. The aim of the present studies was to determine the functional and population diversity of microorganisms present in the tested soil samples, and colonies formed after cultivation, on nutrient agar (NA) and soil extraction agar (SEA) were also analyzed and used for comparison. Besides, the novel self-developed soil extraction agar were used to screen and isolate oligotrophs and physiological, biochemical and plant growth promoting characteristics of them were studied. A total of 7 soil samples were collected from Taichung, Changhua, Nantou, Yunlin and Pingtung. Indigenous microorganisms and colonies grown on NA and SEA were used as inoculants to test for their abilities to utilize 31 carbons using Biolog-EcoPlateTM system , meanwhile 16S rDNA libraries derived from representative samples were constructed and analyzed. In addition, the isolated oligotrophs were characterized for their carbon source utilization, enzymatic activities, antibiotic susceptibility, free-living nitrogen fixation, phosphate solubilization, production of indole-3-acetic acid and aminocyclopropane-1-carboxylate deaminase. The results demonstrated that average well color development values of colonies grown on nutrient-poor SEA were generally higher than that on nutrient-rich NA, and carbon utilization patterns of indigenous microorganisms in most soil samples was more similar to that colonies derived from SEA. Numbers of bacterial genera obtained from colonies grown on SEA were higher than that grown on NA. A total of 76 isolates were obtained from SEA, and they showed facultatively oligotrophic behavior since they can also be cultivated on NA. These isolates belonged to a wide range of bacteria phyla including Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria phyla. Among them, 66 isolates could even grow on nutrient-limited water ager. Most of these isolates belonging to phylum Actinobacteria, Firmicutes and Proteobacteria showed esterase (C4) activities while the abilities to produce α-chymotrypsin and Naphthol-AS- BI-phosphohydrolase can be used to differentiate them. All the tested Gram-positive/negative isolates were sensitive to Imipenem. Regarding their plant growth promoting traits, 24 isolates can grow on nitrogen-free medium and caused color changes of medium. The highest free-living nitrogen fixing activity was recorded in strain OTA1-2F (Pseudomonas aeruginosa). When cultivated in nitrogen-free medium without addition of carbon source, only strain OT03J (Arthrobacter globiformis), OT03C (Arthrobacter niigatensis) and OT03M (Bacillus aryabhattai) showed free-living nitrogen fixing activities. Moreover, 58 isolates can grow on National Botanical Research Institute's phosphate growth medium, and strain OTA1-2I (Pseudomonas jinjuensis) had the highest phosphate solubilizing activity. For all these isolates, there are no detectable phosphate solubilizing activities in NBRIP growth medium when either glucose or mineral salt was removed. A total of 71 isolates can produce indole-3-acetic acid, and the highest quantity was recorded in strain OT03G (Arthrobacter oryzae). Strain OT03E (Arthrobacter pascens) showed indole-3-acetic acid producing activity in nutrient agar containing tryptophan, while strain OT03A (Paenibacillus glycanilyticus) showed activity in soil extraction agar containing tryptophan. Strain OT03J, OT03L (Arthrobacter niigatensis), OT03G, OT03E, OT03F (Arthrobacter pascens), OTA1-2J (Brevibacterium permense) and OTA1-3H (Cupriavidus taiwanensis) had aminocyclopropane-1-carboxylate deaminase activities. Facultative oligotrophs which can utilize a variety of carbon sources and possess plant growth promoting traits may have better environmental adaptabilities, to become more suitable microbial fertilizers.
土壤中養分的分布與含量存在著相當大的變異,此一高度異質性的微域環境孕育許多對養分需求不高、且可適應高度變化的少養分營微生物(oligotroph)。過去在土壤微生物的研究中,主要依賴富含養分的人工培養基針對特定功能設計之篩選培養基,進行目標微生物的分離,對於土壤中的少養分營微生物了解甚為缺乏。本研究探討原土壤與經肉汁培養基和土壤抽出液培養基培養之微生物多樣性,進一步利用土壤抽出液培養基分離少養分營細菌,並針對各分離株之生理生化特性和植物生長促進能力進行瞭解。土樣分別採自台中、彰化、南投、雲林及屏東地區,利用微生物對31種碳源之利用程度代表微生物群落功能多樣性,並利用菌株分離純化及選殖株基因庫之16S rDNA基因庫序列分析代表族群多樣性。此外,各分離株進行碳源利用、基質酵素反應、抗生素感受性、游離固氮活性、溶磷活性、吲哚乙酸生成能力和氨基環丙烷-1-羧酸脫氨酶活性等生理生化和植物生長促進特性之分析。結果顯示土壤抽出液培養基的養分雖然低於肉汁培養基,但大部分土樣於土壤抽出液培養基培養上形成的全部菌落具有較高的平均碳源利用率,且原土樣之微生物所利用的碳源類型與經土壤抽出液培養基形成的所有菌落之碳源利用類型接近,說明土壤抽出液培養基所培養的微生物碳源利用活性與原土樣之微生物表現較為相似。族群多樣性的研究中發現OT11和OTA1-2土樣在土壤抽出液培養基形成全部菌落之細菌菌屬較肉汁培養基形成全部菌落之細菌菌屬各多6和8個,說明土壤抽出液培養基形成所有菌落之微生物群落較能呈現原土樣中的微生物多樣性。研究中以土壤抽出液培養基共分離76個分離株,均可於肉汁培養基上生長,皆屬於兼性少養分營細菌,經16S rDNA序列分析歸類於Actinobacteria、Bacteriodetes、Firmicutes與Proteobacteria細菌門,其中66株可生長於養分含量更少之純水培養基。酵素活性圖譜分析結果顯示Actinobacteria、Firmicutes和Proteobacteria菌門中的少養分營分離株對Esterase(C4)有較多的酵素正反應,另外可藉由α-chymotrypsin和Naphthol-AS- BI-phosphohydrolase酵素的反應活性來區分Actinobacteria、Firmicutes和Proteobacteria三種菌門。6株革蘭氏陽性分離株皆對Piperacillin、Imipenem、Cephalothin、Cefamandole具有感受性,所有試驗的革蘭氏陰性菌也對Imipenem抗生素具有感受性,說明Imipenem可抑制細菌之生長。在植物生長促進特性的研究中,結果顯示24株兼性少養分營分離株可於未添加氮源培養基上產生顏色變化,以OTA1-2F(Pseudomonas aeruginosa)具有最高的游離固氮活性。具有游離固氮活性之OT03J(Arthrobacter globiformis)、OT03C(A. niigatensis)和OT03M(Bacillus aryabhattai)分離株於未添加碳源之培養條件下亦可測得固氮活性,而其他菌株須提供碳源才具有固氮活性。此外,58株菌株可於磷酸三鈣培養基生長,其中OTA1-2I(P. jinjuensis)具有最高的溶磷活性,但於未添加葡萄糖或無機鹽類的環境中,所有試驗之兼性少養分營分離株皆無溶磷活性。71株中有48株具吲哚乙酸生成能力,OT03G (A. oryzae)具有最高的吲哚乙酸生成能力。另外OT03E(A. pascens)僅於含有色氨酸的肉汁培養液中有吲哚乙酸生成能力,但OT03A(Paenibacillus glycanilyticus)則只於含有色氨酸的土壤抽出液中才有吲哚乙酸的生成,說明不同培養基組成會影響吲哚乙酸的生成。OT03J、OT03L(A. niigatensis)、OT03G、OT03E、OT03F(A. pascens)、OTA1-2J(Brevibacterium permense)和OTA1-3H(Cupriavidus taiwanensis)皆可測到氨基環丙烷-1-羧酸脫氨酶活性,具有幫助植物抵抗逆境之潛力。本研究分離之多數兼性少養分營分離株可生長於低養分和高養分培養基,其中許多可利用多種碳源並具有多項植物生長促進特性,可開發為具較佳環境適應性之微生物肥料。
URI: http://hdl.handle.net/11455/90147
文章公開時間: 2015-01-12
顯示於類別:土壤環境科學系

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