风干土壤中氨氧化微生物的恢复

Restoration of microbial ammonia oxidizers in air-dried forest soils upon wetting

【目的】比较历史风干土壤与加水恢复培养土壤中氨氧化古菌AOA和细菌AOB的组成与数量差异,探究风干土壤用于后续微生物生理生态学研究的可能性;明确我国典型酸性森林土壤中,海洋类Group 1.1a是否为数量上占据优势的古菌AOA生态型。【方法】针对中国生态系统研究网络10个台站的典型森林土壤样品,围绕风干保存和加水培养两种处理,通过高通量测序土壤氨氧化古菌及细菌amoA标靶基因,分析氨氧化微生物群落组成的变化规律;利用实时荧光定量PCR和DGGE指纹图谱技术,研究森林土壤微生物群落16S rRNA基因的数量变化规律,以及氨氧化细菌和古菌群落结构的差异。【结果】10个历史风干土壤加水培养28天后,土壤细菌和古菌数量均急剧增加,最高可达3230倍和568倍;其中8个土壤中氨氧化古菌AOA明显增加,5个土壤中氨氧化细菌AOB表现出明显的增加趋势。然而,高通量测序和系统发育分析表明,历史风干土壤与加水恢复培养土壤中AOA和AOB的群落组成无明显变化。Group 1.1b是氨氧化古菌的优势类群,而氨氧化细菌的主要类群是Nitrosospira螺菌属。氨氧化古菌和细菌的比例与总氮浓度呈显著正相关(r2=0.54,P<0.05),表明酸性条件下土壤矿化并提供铵态氮底物可能是古菌氨氧化的驱动机制。【结论】风干土壤加水恢复培养后,AOA和AOB的种群数量大多出现增加的趋势,但其物种组成未发生显著变化,表明风干保存的土壤样品可用于后续室内培养,开展微生物生理生态学研究。与已有的海洋AOA生态型主导酸性土壤氨氧化类群的报道不同,土壤Group 1.1b是本研究森林土壤中的优势类群。

[ Objective] This study was aimed to investigate the abundance and community shift of ammonia-oxidizing archaea （AOA） and bacteria （AOB） in air-dried forest soils in response to water addition, to explore the applicability of air-dried soil for microbial ecology study, and to elucidate whether AOA within the marine group 1. 1a dominate ammonia oxidizers communities in the acidic forest soils in China. [Methods] Soil samples were collected from 10 forest sites of the China Ecosystem Research Network （CERN） and kept under air-drying conditions in 2010. In 2013 the air-dried soil samples were adjusted to 60% of soil maximum water holding capacity for a 28-day incubation at 28 ℃ in darkness. DGGE fingerprinting, clone library construction, pyrosequencing and quantitative PCR of amoA genes were performed to assess community change of ammonia oxidizers in air-dried and re-wetted soils. [ Results] After incubation for 28 days, the abundance of bacteria and archaea increased significantly, up to 3,230 and 568 times, respectively. AOA increased significantly in 8 samples, and AOB increased significantly in 5 of 10 samples. However, pyrosequencing of amoA genes reveals insignificant changes in composition of AOA and AOB communities. Phylogenetic analysis of amoA genes indicates that archaeal ammonia oxidizers were predominated by AOA within the soil group 1. 1b lineage, while the Nitrosospira-like AOB dominate bacteria ammonia oxidizer communities. There was a significantly positive correlation between AOA/AOB ratio and total nitrogen （ r^2 = 0. 54, P 〈 0.05 ） , implying that soil ammonia oxidation might be dominated by AOA in association with ammonium released from soil mineralization. [ Conclusion ] Phylogenetic analysis suggest that AOA members within the soil group 1. 1 b lineage were not restricted to non-acidic soils as previously thought. The abundance rather than composition of AOA and AOB changed in response to water addition. This indicates that air-dried soil could be of help for microbial hiogeography study.