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题名: 水稻土中微生物群落和厌氧铵氧化细菌的分子生态学研究
作者: 白 刃1
学位类别: 博士
答辩日期: 2015-11
授予单位: 中国科学院生态环境研究中心
授予地点: 北京
导师: 贺纪正 ; 张丽梅
关键词: 水稻土 ; 功能基因微阵列 ; functional gene array ; 厌氧铵氧化 ; anammox ; 稳定性同位素示踪技术 ; stable isotope tracertechnique ; 剖面分布 ; soil profile ; Paddy soil
其他题名: Molecular ecological studies of microbial community and anammox bacteria in selected paddy soils
学位专业: 生态学
中文摘要: 征进行了深入研究,为揭示水稻田土壤氮素转化特征和机制提供了重要参考。 论文取得的主要研究结果如下: (1)三种代表性水稻土剖面微生物的群落结构:对取自江苏滨海(始成 土)、广东雷州(氧化土)和湖南桃源(老成土)长期种植水稻的土壤于 50*70 cm 的聚乙烯桶中建立中型培养土柱,于温室中淹水-落干过冬,并于次 年按田间管理方式进行施肥及水稻栽培,于水稻的分蘖期和抽穗期沿0-5 cm, 5-20 cm,20-40 cm 和40-60 cm 四个剖面深度上进行样本采集。利用Illumina Miseq 高通量测序技术对土样中细菌和古菌的16S rRNA 基因分析结果表明,土 壤细菌和古菌的群落结构在不同类型的水稻土以及土壤剖面梯度上差异显著, 但在不同水稻生长期之间差异不明显。同时剖面梯度上微生物的群落结构差异 在0-5 cm 和5-60 cm 的剖面之间最为显著,而在5-60 cm 的三个剖面之间微生 物群落结构的差异相对较小。CCA 和VPA 结果显示,土壤环境因子对土壤细 菌和古菌群落结构变异的解释量分别为55.6%和76.1%,土壤pH、碳氮比和有 机碳等土壤化学性质显著影响土壤细菌和古菌的群落组成。 (2)三种代表性水稻土剖面微生物的功能结构:利用功能基因微阵列技术 对以上取自三种水稻土中型培养土柱的剖面样品中土壤微生物的功能结构进行 了分析。结果表明,微生物功能基因的多样性和丰度总体上在滨海始成土和桃 源老成土中显著高于雷州的氧化土;同时,土壤微生物的功能结构在不同类型的水稻土以及不同水稻生长期之间差异显著,但在水稻土的土壤剖面梯度上变 化不明显。CCA 和VPA 结果显示,土壤环境因素对土壤微生物功能结构变异 的解释量为52.4%,同时土壤pH、盐度和有机碳等环境因子显著影响土壤微生 物的功能结构。通过分析和碳、氮、磷、硫四种元素转化相关的功能基因信号 变化,发现这些功能基因的群落结构也在土壤类型之间存在明显差异,且绝大 多数与碳和氮循环相关的功能基因在水稻抽穗期显著上调。 (3)不同水稻土中厌氧铵氧化细菌的多样性:利用16S rRNA 基因的PCR 扩增-克隆和测序方法,对取自我国10 个水稻种植区的大田土壤剖面样品( 0- 20 cm,20-40 cm,40-60 cm 和60-80 cm)中厌氧铵氧化细菌的群落结构和丰度 进行了研究。结果表明,检测到的厌氧铵氧化细菌全部为Candidatus Brocadia 属和Candidatus Kuenenia,同时Candidatus Brocadia 属占到了绝对优势,并且 Candidatus Brocadia 属有着更广的分布范围。结合环境因子分析显示,土壤pH 和铵离子浓度是影响厌氧铵氧化细菌群落结构的重要驱动因子,它们对厌氧铵 氧化细菌群落结构的解释量分别为22.2%和27.8%。 (4)不同水稻土中厌氧铵氧化细菌的活性、丰度和群落结构:通过稳定性 同位素示踪技术和荧光定量PCR 以及克隆测序等方法,研究了温室培养的三种 水稻土中型土柱剖面上厌氧铵氧化细菌的活性、丰度及群落结构。研究发现厌 氧铵氧化速率在0.02-0.77 nmol N g-1 dry soil h-1 的范围内,对水稻土中氮气生成 的贡献率为0.4%-12.2%。厌氧铵氧化细菌的活性、丰度和多样性在中性和碱性 水稻土高于酸性水稻土,同时在深层(5-60 cm)水稻土高于表层(0-5 cm)水 稻土。同大田样品研究结果类似,三种水稻土中厌氧铵氧化细菌以Candidatus Brocadia,Candidatus Kuenenia 为主,部分样品检测到Candidatus Jettenia,且 其群落组成在三种类型的水稻土中差异显著。相关性分析表明厌氧铵氧化细菌 的活性及丰度与土壤pH、盐度及氧化还原电位等由土壤母质所决定的环境因子 显著相关,并且沿着水稻土垂直剖面梯度变化的溶解氧浓度也是影响厌氧铵氧 化过程的一个重要限制性因子。
英文摘要: Microbes perform versatile metabolisms and mediate many essential biological and ecological processes. Paddy rice fields occupy large agricultural areas of China, and cover diverse soil types. Due to the limitation of existing research approaches, the mechanisms of how diverse soil properties which were caused by different parent materials influence the microbial community and functional structures of the oaddy soils are unclear. Furthermore, the flooding-drought alternation in paddy soils produces their unique oxidation-reduction (redox) conditions, while how these conditions determine soil microbial communities and biogeochemical cycling is also essential to understand nutrient cycling in paddy soils and its ecological and environmental significance. High throughput sequencing and Geochip technologies are the powerful techniques to investigate soil microbial communities and functions. By combining stable isotope tracer technique with these technologies, we could further study the microbial mechanisms of ecological processes in paddy soils. Hence, we studied the microbial community and functional structure along profiles in three typical paddy soils by utilizing Illumina Miseq high throughput sequencing and functional gene array (FGA), and analyzed the effects of soil properties and environmental attributes on microbial community and functional structures. In addition, we also investigated the community composition of anaerobic ammonium oxidation (anammox) bacteria in paddy fields through large-scale sampling, and studied their activity and abundance along profiles in different paddy soils. The main results of these studies are as follow: 1) Microbial community structure along profiles in three typical paddy soils: we incubated paddy soils sample from Binhai (BH) of Jiangsu Province (inceptisol), Leizhou (LZ) of Guangdong Province (oxisol) and Taoyuan (TY) of Hunan Province (ultisol) in polyethylene columns (50 cm in diameter and 70 cm in height) in a greenhouse. After flooding-drought management over a winter, the soils received watering, fertilization and rice plantation as in normal fields practices in the following year. Soil samples were collected at tillering stage and heading stage of the rice plants along soil profiles (0-5 cm, 5-20 cm, 20-40 cm, 40-60 cm). 16S rRNA genes of soil bacterial and archaeal were analyzed by using Illumina Miseq sequencing. Results indicated that community structures of the soil bacteria and archaea were greatly variable among soil types and along soil profiles, but were similar between growth stages of the paddy rice. Meanwhile, the variation of microbial community structures mainly presented between top soils layers (0-5 cm) and sub-soils layers (20-60 cm). By conducting CCA and VPA, it further indicated that environmental attributes explained 55.6% and 76.1% of the variation of soil bacterial and archaeal communities respectively, and soil properties such as pH, carbon-nitrogen ratio and total organic carbon significantly influence community composition of soil bacteria and archaea. 2) Functional structure of the microbial communities along profiles in three typical paddy soils: we studied the functional structure of soil microbes in the same soil samples as above by using FGA. Results indicated that diversity and abundance of microbial functional genes in BH and LZ soils were significantly higher than in TY soils. Functional structure varied among soil types and between rice growth stages, but were similar among soil profiles. By conducting CCA and VPA analyses, it further indicated that environmental attributes explained 52.4% of the variation of soil microbial functional structure. Soil properties such as pH, salinity and total organic carbon significantly effected community structures of microbial functional genes. We also focused on the pattern of genes involved in carbon, nitrogen, phosphorus and sulfur metabolisms, and found that community structures of these genes also varied among soil types. Most of the genes participated in carbon and nitrogen cycling increased significantly in the heading stage of rice growth. 3) Diversity of anammox bacteria in different paddy soils: we studied diversity, abundance and community structure of anammox bacteria in the profiles (0-20 cm, 20-40 cm,40-60 cm and 60-80 cm) of selected Chinese paddy soils by utilizing PCR, cloning and sequencing of anammox bacterial 16S rRNA genes. Results showed that all the anammox bacteiral sequences we retrieved belonged to Candidatus Brocadia and Candidatus Kuenenia, and Candidatus Brocadia was more predominant. CCA results showed that soil pH and ammonium concentration significantly influenced anammox bacterial community composition, and explained the 22.2% and 27.8% of the variation of the community composition. 4) Anammox bacterial activity, abundance and community structure: by utilizing stable isotope tracer technique, real-time PCR, cloning and sequencing, we investigated anammox bacterial activity, abundance and community along profiles in three paddy soils. Results showed that rates of anammox were within the range of 0.02-0.77 nmol N g-1 dry soil h-1, and anammox contributed 0.4%-12.2% of the total N2 production. Also, anammox bacterial activity, abundance and diversity were higher in alkaline and neutral paddy soils than in acidic soils, and were higher in subsoils (20-60 cm) than in top soils (0-5 cm). Similar to the results from the field study, Candidatus Brocadia and Candidatus Kuenenia were the dominant groups in these soils, and Candidatus Jettenia could also be detected in some samples. In addition, community composition was significantly different from each other in the three paddy soils. Correlation analysis indicated that activity and abundance of anammox bacteria significantly correlated to soil pH, salinity and redox, which were largely determined by soil parent materials, and the fluctuating concentrations of dissolved oxygen along profiles were also a limiting factor to anammox process in paddy soils.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/38599
Appears in Collections:中澳联合土壤环境研究室_学位论文

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作者单位: 1.中国科学院生态环境研究中心

Recommended Citation:
白 刃. 水稻土中微生物群落和厌氧铵氧化细菌的分子生态学研究[D]. 北京. 中国科学院生态环境研究中心. 2015.
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