福山森林土壤微生物族群、生質量、功能和基因多樣性

Microbial Population, Biomass, Functional and Genetic Diversity of Fushan Forest Soils

蔡書憲(Shu-Hsien Tsai)

指導教授 ： 楊盛行

國立臺灣大學/生命科學院/微生物與生化學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.02532

摘要

福山森林為台灣長期生態研究樣區之一，為探討福山森林不同地形之土壤微生物生態，本研究選定福山地區溪谷、中坡及稜線等不同地形位置，於不同季節測定其溫度和日照等環境條件，水分含量、pH值、總有機碳、總氮和C/N比等土壤性質，測定細菌、真菌、放線菌、纖維素分解菌、溶磷菌及固氮菌含量及微生物生質碳和氮等微生物指標。並以16S rDNA序列對細菌分離株進行初步鑑定，利用PCR-DGGE技術及建立16S rDNA基因選殖庫，探討不同地形位置細菌基因多樣性和使用BIOLOG® Eco-MicroPlates及纖維素分解酵素、聚木醣分解酵素、幾丁質分解酵素及酸性磷酸酯解酵素等四種土壤酵素研究微生物功能多樣性。結果顯示，福山森林土壤水分含量、總有機碳含量和總氮含量，以稜線最高，其次為中坡和溪谷最低。pH值則是溪谷>中坡>稜線。不同地形位置微生物菌數及微生物生質量含量為稜線>中坡>溪谷，並且受到降雨型態和溫度的影響，冬季具有較低的數值。福山土壤剖面pH值以OA層最低，隨著土壤深度增加而增加，總有機碳含量、總氮含量、微生物菌數及微生物生質量則隨著土壤深度增加而減少。微生物生質量和非生物因子具有較佳之相關性，為較佳的微生物指標。功能多樣性方面，不同樣區利用BIOLOG® Eco-MicroPlates所得的碳源利用數量以溪谷地區最高，與土壤pH有關，四種土壤酵素活性則皆以溪谷地區具有比較低的酵素活性。台灣福山森林土壤所分離出的細菌，若以屬作為分類基本單位，可以分成革蘭氏陽性的Bacillus、和Streptomyces以及革蘭氏陰性菌Brevundimonas、Burkholderia、Chromobacterium、Flavobacterium、Pseudomonas和 Ralstonia等菌屬。土壤DNA萃取與純化方面，比較五種不同萃取土壤DNA方法，以方法E萃取森林土壤DNA具最佳產率和純度。PCR-DGGE分析結果顯示在不同的樣區中皆存在相似的主要條帶，而不同樣區所呈現的PCR-DGGE圖譜，主要的差異在次要的條帶，不同的地形位置會影響微生物菌數及多樣性。九個16S rDNA 選殖基因庫，總共包含Proteobacteria、Acidobacteria、Actinobacteria、Bacteroidetes、Cyanobacteria、Firmicutes、Gemmatimonadetes、Nitrospirae、Planctomycetes、candidate division TM7 和 Verrucomicrobia等不同的族群。其中Proteobacteria、Acidobacteria和Actinobacteria分別佔全部選殖基因庫的 49.1%、32.3% 和6.3%，而其他的族群則皆未超過 6%。不同地形位置中稜線地區含有最高細菌多樣性，有機質層和深層土壤相比亦具有較高細菌多樣性。
總結來說，本研究建立台灣福山森林土壤性質及微生物生態背景資料外，並提供未來長期研究的依據，由相關性分析顯示微生物生質量可用於福山森林土壤微生物生態長期偵測微生物指標之工具，地形及土壤深度對於細菌的分佈不僅會產生數量上的差異，也會造成功能及基因多樣性的改變，因此在進行長期偵測時必須考慮地形、季節和土壤深度等變因所帶來的影響。固氮菌屬(Burkholderia、Delftia、Frankia、Herbaspirillum、Methylocystis和Pleomorphomonas)、銨氧化菌屬(Nitrosococcus)及甲烷氧化菌屬(Methylobacter、Methylocystis和Methylocapsa)可作為未來功能性基因 (nifH、amoA及pmoA)研究之目標。

關鍵字

森林土壤 ； 微生物生態 ； 16S rDNA選殖基因庫 ； 變性梯度凝膠電泳 ； 土壤酵素 ； 地形

並列摘要

Fushan forest is one of the Long Term Ecological Research (LTER) sites. To investigate the effect of topography on microbial ecology of Fushan forest, valley, middle-slope and ridge were selected. Environmental conditions (soil temperature and light intensity); soil properties (moisture content, pH, total organic carbon, total nitrogen and C/N ratio); populations of bacteria, actinomycetes, fungi, cellulolytic, phosphate-solubilizing, nitrogen-fixing microbes, and microbial biomass (carbon and nitrogen) were measured at different seasons. In addition, experiments were carried out to identify some bacterial isolates by 16S rDNA, to analyze bacterial diversity of different topographies by PCR-DGGE and 16S rDNA clone library, to study the functional diversity using BIOLOG® Eco-MicroPlates and to investigate the soil enzyme (cellulase, xylanase, chitinase, and acidphosphatase) activities. The results showed that moisture content, total organic carbon and total nitrogen were the highest in the ridge soils, followed by the middle-slope and the valley was the lowest, but the pH was reversed. Microbial populations and biomass contents were the highest in the ridge, then in the middle-slope, and the valley was the lowest. Because of rainfall patterns and temperature effect, the microbial populations and biomass were the lowest in winter season. In the soil profiles, the pH was the lowest at OA horizon and increased with soil depths, while total organic carbon, total nitrogen, microbial populations, and microbial biomass were reversed. Compared to microbial populations, microbial biomass have higher correlation with abiotic factors, so it was the best microbial indicator. Substrate richness (S), obtained from BIOLOG analysis of valley, middle-slope and ridge soil samples were the generally highest in valley due to the higher pH. The valley soil had the lowest soil enzyme activities. The Fushan forest soil bacterial isolates were included Bacillus, Streptomyces, Brevundimonas, Burkholderia, Chromobacterium, Flavobacterium, Pseudomonas and Ralstonia. Five different methods (A to E) were used to extract soil genomic DNA and one method (method E) was found to be suitable in terms of quantity and quality of DNA. Analysis using denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplicons of 16S rDNA showed that the bacterial diversity of the three sites were very similar to each other in the major bands and the variation was omly in the minor bands. Topography influenced the quantity and diversity of microbial populations. Phylogenetic analysis revealed that the clones from nine clone libraries were represented Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, candidate division TM7 and Verrucomicrobia. Members of Proteobacteria, Acidobacteria and Actinobacteria constituted 49.1%, 32.3% and 6.3% of the clone library, respectively, whereas the remaining bacterial divisions each comprised less than 6%. The ridge site exhibited the highest bacterial diversity among the tested sites indicating the influence of topography. Bacterial composition was more diverse in organic layer than that in deeper horizons.
In conclusion, this study documented the soil properties and microbial ecology background data in Fushan forest of Taiwan. It provides the reference to further long term ecological research. According to the correlation analysis, microbial biomass is the suitable microbial indicator to monitor the microbial ecology in Fushan forest soils. Topography and soil depth influenced the microbial populations, functional and genetic diversities. It have to be considered that the effect of topography, season and soil depth, when the long term monitor want to be process. In this study, there are some functional bacterial genus were determined, included nitrogen-fixing bacteria (Burkholderia, Delftia, Frankia, Herbaspirillum, Methylocystis and Pleomorphomonas), ammonium oxidizing bacteria (Nitrosococcus), and methane oxidizing bacteria genus (Methylobacter, Methylocystis and Methylocapsa). Their functional genes (nifH, amoA and pmoA) can provide microbial ecology information in the future.

並列關鍵字

16S rDNA clone library ； DGGE ； forest soil ； microbial ecology ； soil enzymes ； topography

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