以穩定同位素探針探討水田與旱田之土壤硝化作用及其活性微生物族群結構差異

Abstract

農田的管理與利用，使其形成獨特的生態系統，透過不同的灌溉、施肥方式與種植作物，對其生態系統的可持續性和功能產生不同程度的影響，微生物硝化作用在土壤生態系統的氮循環中發揮著重要作用，硝化作用由氨氧化古菌 (ammonia-oxidizing archaea, AOA) 、氨氧化細菌 (ammonia oxidizing bacteria, AOB) 執行氨氧化反應，然而氨氧化菌在土壤之自營硝化作用中與溫室氣體及環境因子的相對重要性仍不清楚。 本研究選擇了霧峰農業試驗所之水稻與甘藍輪作的農田土壤做為水、旱田代表，探討兩種土壤經由基肥與追肥後，活性氨氧化群落的結構變化與氧化亞氮 (N2O) 在其土壤中的排放潛力，以及與環境因子之間的相關性。 由尿素培養的過程中，顯示出四種土壤在42天內的硝化活性變化，分別為水稻基肥 (Rice-B) 4.37、水稻追肥 (Rice-T) 3.50、甘藍基肥 (Cabbage-B) 1.81與甘藍追肥 (Cabbage-T) 0.57 µg NO3- -N g-1 soil day-1。氧化亞氮 (N2O) 於四種土壤中的排放潛力皆低於其能被偵測的極限，因此未有N2¬O排放被測得。amoA基因的實時定量PCR和16S rRNA基因的Illumina MiSeq定序顯示，AOA群落具有不同程度的增加，AOB則僅於Rice-B與Cabbage-B展現相對較小的活性反應，表示在四種土壤中由AOA主導氨氧化的反應過程。 透過DNA穩定同位素探針 (DNA stable-isotope probing, DNA-SIP) 進一步表示，由13CO2所標記的四種土壤中，Rice-B與Cabbage-B土壤之活性AOA在數值上比其AOB高出1.32與42.69倍，而Rice-T與Cabbage-T則僅出現對AOA的活性標記，並且在乙炔 (C2H2) 的加入後，完全消除氨氧化菌群對13CO2的同化作用，根據系統發育分析表示，古菌氨氧化主要由土壤group 1.1b之54d9 cluster與29i4 cluster的AOA催化，Nitrosospira cluster 3-like AOB則僅於水稻與甘藍農田的基肥土壤中出現，透過冗餘分析研究顯示四種土壤的氨氧化作用主要受到NO3-、TDN、pH的調控。 這項結果提供在人為干擾的農田土壤環境中，氨氧化活性群落的反應、結構分布與其生理差異的理解。

The management and utilization of agricultural fields create unique ecosystems, and different irrigation, fertilization methods, and crop choices can have varying impacts on the sustainability and functionality of these ecosystems. Microbial nitrification plays a crucial role in the nitrogen cycle of soil ecosystems. Nitrification is performed by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) through the process of ammonia oxidation. However, the relative importance of ammonia oxidizers in soil's autotrophic nitrification, in relation to greenhouse gases and environmental factors, remains unclear. This study selected rice paddy and cabbage under rotation at the Taiwan Agricultural Research Institute as representative examples of rice paddy and upland farmland,respectively. The aim was to investigate the structural changes in the active ammonia oxidizer communities and the potential emissions of nitrous oxide (N2O) in these soils after basal and additional fertilization. Additionally, the study aimed to assess the correlation between these factors and environmental variables. In urea-amended microcosms, the four types of soils exhibited varying levels of nitrification activity within 42 days. The nitrification rates, expressed as 4.37, 3.50, 1.81 and 0.57 µg NO3--N g-1 soil per day in the Rice-B (rice basal fertilization), Rice-T (rice topdressing), Cabbage-B (cabbage basal fertilization) and Cabbage-T (cabbage topdressing) soils. The emission potential of nitrous oxide (N2O) in all four soils was found to be below the detectable limit, and thus, no N2O emissions were measured. Real-time quantitative PCR of the amoA gene and Illumina MiSeq sequencing of the 16S rRNA genes revealed varying degrees of increase in the AOA communities. AOB, on the other hand, showed relatively minor activity responses only in Rice-B and Cabbage-B, indicating that AOA dominated the ammonia oxidation process in the four soil types. Further analysis using DNA stable isotope probing (DNA-SIP) revealed that in the four soils labeled with 13CO2, the active AOA in Rice-B and Cabbage-B soils showed 1.32-fold and 42.69-fold higher values, respectively, compared to their AOB counterparts. In contrast, Rice-T and Cabbage-T only exhibited activity labeling for AOA, and the addition of acetylene completely abolished the assimilation of 13CO2 by ammonia oxidizer populations. Phylogenetic analysis suggested that archaeal ammonia oxidation was predominantly catalyzed by soil fosmid 29i4-related AOA within the soil group 1.1b lineage. Nitrosospira cluster 3-like AOB, on the other hand, were only detected in the basal fertilized soils of rice and cabbage fields. Redundancy analysis demonstrated that the ammonia oxidizers in the four soils were mainly regulated by NO3-, TDN, and pH. This finding provides insights into the response, structural distribution, and physiological differences of the ammonia oxidizer communities in agriculturally disturbed soil environments.