Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90146
標題: Isolation, trophic and plant growth promoting characterization of facultative oligotrophs
兼性少養分營細菌之分離、營養及植物生長促進特性研究
作者: Yu-Shiuan Huang
黃郁瑄
關鍵字: oligotrophs;methanol utilization;agarolytic bacteria;CO2 absorbent;bacteriochlorophyll;plant growth promoting traits;少養分營微生物;甲醇利用;洋菜分解細菌;二氧化碳吸附劑;細菌葉綠素;植物生長促進特性
摘要: 
Oligotrophs, a group of bacteria which get adapted to grow under conditions of C/nutrient starvation present in a wide range of microhabitats. Nutrients in soils were heterogeneously distributed and quantities were unlike the cultural media generally used in microbiological laboratory. Owing to the selective isolation of rapidly growing bacteria by using conventional cultivation methods, the exploration of oligotrophs seems limited. The present studies were undertaken to perform a preliminary isolation, trophic and plant growth promoting (PGP) characterization of oligotrophs from a variety of soils. A total of four oligotrophic sites were selected and used for bacterial isolation. These includes two coastal sandy soils from Miaoli and Pingtung, one wetland soil from Taichung and one red soil from Miaoli. Samples were serial diluted and plated on 1,000-fold-diluted nutrient agar to screen and isolate oligotrophs. Phylogenetic analyses of these oligotrophic isolates were performed based on their 16S rDNA sequences. To clarify trophic characteristics of oligotrophs, growths of these isolates were determined on nutrient agar, methanol containing agar and water agar. Requirements of CO2 or light for the growth of these oligotrophic isolates were evaluated to study their autotrophic behavior. Genotypic characterizations were also conducted using PCR and primer sets specific for target genes. Besides, the PGP potentials regarding nitrogen fixation, phosphate solubilization and seed germination of these oligotrophic isolates were examined. A total of 74 isolates with different colony morphology were selected and purified. They are widely distributed in three bacterial phyla, five -classes and 23 -genera. These isolates were considered as facultative oligotrophs since they can also form colonies on nutrient agar. 69 isolates shared higher sequence similarities (> 97%) of 16S rDNA sequence with their most closely related type strains. In addition, five isolates namely S-NB-12 and S-NB-16 shared lower sequence similarity (95.37%) with Roseomonas rosea 173-96T while K-NB-13, R-NB-9 and W-NaCl-5 isolated from different soil samples shared lower sequence similarity (96.41%) with Sphingopyxis wooponensis 03SU3-PT. These isolates may represent novel bacterial species. The oligotrophic behavior of the genus Bacillus, Cellulomonas, Gordonia, Methylobacterium, Methylophilus, Mycobacterium, Paracoccus, Pseudomonas and Sinomonas consisting of 24 isolates have not been reported before. Some of these isolates (13/74) can use methanol as their heterotrophic carbon source. Utilization of methanol and presence of mxaF gene were demonstrated in isolates belonging to the genus Aureimonas . Many isolates (36/74) can grow on water agar, but only 17 isolates showed agar utilization as evidenced by the appearance of clear zone around colonies after stained with Lugol's solution. The unique agarolytic characteristic was found in isolates belonging to the genus Arthrobacter, Cellulomonas, Methylobacterium, Methylophilus, Nocardioides, Rhizobium, Sinomonas and Sphingopyxis. Growth of six isolates namely Aeromicrobium sp. W-NaCl-1、Mycobacterium sp. K-NB-9、Mycobacterium sp. K-NB-11、Porphyrobacter sp. K-NB-10、Terrabacter sp. R-NB-10 and Yangia sp. K-NaCl-3 was effected by CO2, as evidenced when CO2 absorbent was placed in the chamber to produce a CO2-limited condition. The puf genes which encode the photosynthetic reaction center type II structural proteins were obtained from Methylobacterium sp. W-NB-14 and Porphyrobacter sp. K-NB-10. When cultivation in dark condition, these two isolates showed less colony formation. Regarding PGP traits, the free-living nitrogen fixation was not found in all these facultative oligotrophs. More than half of these isolates (39/74) could solubilize tricalcium phosphate no matter glucose or sucrose was used as carbon source. The highest phosphate solubilizing activity was recorded as 322 ppm in Bacillus sp. R-NB-5. Inoculation of Aureimonas sp. S-NaCl-1 or Brevundimonas sp. S-NaCl-7 stimulate the growth of radical, epicotyl length and germination rate of Lettuce (Dahu 366). The present work provided preliminary studies on the phylogeny, trophic and plant growth promoting characteristics of facultative oligotrophs. The trophic behavior of these isolates may give them advantages to survive and adapt to various environmental conditions, which will serve as superior candidates used in agriculture.

少養分營微生物(oligotrophs)廣泛存在自然界中,是能在低營養(碳源)培養條件下生長的微生物。微生物棲息之微域環境(niche)中養分分布不均且有效性碳含量亦不如微生物培養基充足,又篩選環境微生物易獲得生長速率較快的一群,使得過去與少養分營微生物相關之研究有限。本研究旨在分離土壤少養分營微生物,探討可培養分離株之族群多樣性,並分析其營養與植物生長促進特性。首先從台中高美濕地、苗栗苑裡地區、苗栗通霄海水域場及屏東墾丁白沙灣共採集三個砂土與一個紅土作為分離微生物之土樣。選用稀釋1,000倍肉湯抽出物培養基進行土壤懸浮液之連續稀釋塗抹,分離純化土樣中的少養分營微生物,並利用聚合?連鎖反應與核酸定序技術,進行各分離株之16S rDNA親緣分析。同時,本研究設計肉湯抽出物培養基、甲醇培養基及高純度洋菜培養基對分離株進行異營性培養試驗,另探討二氧化碳與光照對分離株自營生長之影響,並藉由基因型分析結果進一步討論分離株之營養特性。此外,透過固氮、溶磷及種子生物分析測試分離株之植物生長促進潛力。本研究從四個土樣中共分離廣泛分布於3種菌門(phylum)、5種菌綱(class)及24種菌屬(genus)之74株少養分營微生物。其中69個分離株之16S rDNA序列與最相近之標準菌株相似度高於97%。其餘從海水浴場砂土分離之S-NB-12、S-NB-16與Roseomonas rosea 173-96T相似度僅為95.37%,而從不同土樣分離之K-NB-13、R-NB-9、W-NaCl-5與Sphingopyxis wooponensis 03SU3-PT相似度為96.41%,顯示為本土分離之新菌種。全部分離株皆可於未稀釋肉湯抽出物培養基上生長,是可適應廣泛碳源濃度之兼性少養分營微生物。且歸類於Bacillus、Cellulomonas、Gordonia、Methylobacterium、Methylophilus、Mycobacterium、Paracoccus、Pseudomonas及Sinomonas菌屬的24個分離株皆未曾報導過具有少養分營特性,。甲醇利用分析結果顯示部分分離株(13/74)具利用甲醇作為碳源之異營特性,其中Aureimonas菌屬之分離株能利用甲醇生長並增殖出甲醇去氫?基因(mxaF),為本研究之新發現。有近半數(36/74)的分離株可於高純度洋菜培養基上生長,但碘液染色後僅有17個分離株有利用洋菜作為碳源之異營特性,其中歸類於Arthrobacter、Cellulomonas、Methylobacterium、Methylophilus、Nocardioides、Rhizobium、Sinomonas及Sphingopyxis菌屬的分離株可利用洋菜也是新發現之一。將分離株置於含二氧化碳吸附劑之裝置中培養,僅Aeromicrobium sp. W-NaCl-1、Mycobacterium sp. K-NB-9、Mycobacterium sp. K-NB-11、Porphyrobacter sp. K-NB-10、Terrabacter sp. R-NB-10及Yangia sp. K-NaCl-3之菌落生長受到影響,其中文獻未報導過Aeromicrobium、Porphyrobacter及Yangia菌屬具利用二氧化碳作為碳源之自營特性。光能利用分析中僅觀察到Erythrobacter sp. K-NB-18、Erythrobacter sp. W-NB-2、Methylobacterium sp. W-NB-14及Porphyrobacter sp. K-NB-10於稀釋培養基內之菌落生長受到避光處理影響,同時也偵測到Methylobacterium sp. W-NB-14、Porphyrobacter sp. K-NB-10具備光能反應中心基因(pufLM),顯示其於低養分條件下可利用光能補充能源。在植物生長促進特性之測試結果中,顯示全部分離株皆無固氮活性;超過半數的分離株(39/74)於葡萄糖或蔗糖作為碳源之磷酸三鈣培養基內具有溶磷活性,其中又以Bacillus sp. R-NB-5之溶磷活性最佳;Aureimonas sp. S-NaCl-1與Brevundimonas sp. S-NaCl-7之10X、100X、500X、1,000X、2,000X稀釋倍率培養液與對照組相比皆可促進結球萵苣種子(大湖366)之胚根與胚莖生長達顯著差異並能提升發芽率,顯示具有促進植物生長之應用潛力。本研究成功自四種土樣中分離純化共74株兼性少養分營微生物,並對分離株利用甲醇、洋菜、二氧化碳、光能之各種少養分營養特性有進一步的了解。此外部分分離株具植物生長促進之應用潛力,又其於養分分布不均的土壤環境中有生存優勢,具開發為農用微生物製劑之價值。
URI: http://hdl.handle.net/11455/90146
Rights: 同意授權瀏覽/列印電子全文服務,2014-08-31起公開。
Appears in Collections:土壤環境科學系

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