【作者】 刘桂婷；

【导师】 赵其国； 曲东；

【作者基本信息】 西北农林科技大学 ， 环境科学， 2010， 硕士

【摘要】 黄土高原地区为中国古老的雨养农业区,耕地面积为0.18亿公顷,占全国耕地面积的19%,因此对黄土高原的进一步研究具有十分重要的意义。由于侵蚀与干旱的影响,黄土高原区粮食的产量一般低于2.0t/hm2,为全国土壤生产力的低产水平。尽管施用化学氮肥是提高作物产量的必要措施,但长期大量施用能引起硝态氮在根区以下土层的无效累积。有研究表明,由于大量施用氮肥,黄土区的农田土壤剖面已经出现了明显的硝态氮积累现象,土壤中硝态氮的高量累积不仅造成氮肥的浪费和土壤氮素肥力的降低,而且也可能污染地下水。氨氧化作用是硝化作用的第一个反应步骤,也是限速步骤,是全球氮素循环的中心环节。因此,研究氨氧化微生物(细菌和古菌)具有重要的环境意义。研究土壤中氨氧化细菌和氨氧化古菌群落多样性及其结构变化,将为了解土壤硝化作用的特征提供必要的理论依据。本试验样品采集于中国科学院长武农业生态站长期定位施肥基地。试验采用完全随机区组设计方案,设置5个处理：①CK(无肥区)、②M、③NM、④PM、⑤NPM。各处理每种肥料的施入量为：有机肥(牛粪)7500 kg/hm2,N肥(尿素)120 kg/hm2,P肥(过磷酸钙)60 kg/hm2。采用直接法提取土样微生物总DNA,分别采用amoA和Arch-amoA引物扩增基因片段,建立氨氧化细菌和氨氧化古菌的克隆文库,用限制性内切酶进行PCR-RFLP分析。本研究的主要结果如下：1.通过对5种施肥制度下氨氧化细菌amoA基因群落多样性的分析表明：(1)5种施肥处理分别得到59/150、56/150、83/150、71/150、42150个RFLP酶切类型。库容值分别为80.67%、80.67%、60.00%、66.67%和84.56%。各处理均出现了明显的优势菌群。(2)α多样性指数表明不同施肥处理的多样性存在差异,NM处理多样性最高,NPM处理多样性最低。Shannon-Wiener指数(H)与物种丰富度指数(E)数值上均为NM>PM>CK>M>NPM,优势度Simpson指数(Ds)数值上为NM>CK>PM>NPM>M, Margalef指数(dMa)为PM>NM>CK>M>NPM。p多样性指数表明M与NPM之间Sorenson指数为0.61,群落相似性最高；NM、PM与CK之间Sorenson指数均为0.15,群落相似性最低；其余均介于0.23-0.38。表明长期施肥改变了氨氧化细菌的多样性。(3)基于amoA氨基酸序列建立的系统进化树表明,各处理均以Nitrosospira cluster3为优势种群,但不同处理间属于Nitrosospira cluster 3a和3b中克隆子所占的比例大小不同,同时发现有Nitrosospira cluster 4种属。说明不同的土壤肥力环境对氨氧化细菌种属的长期选择导致种属差异。2.通过对氨氧化古菌amoA基因群落多样性的分析表明：(1)CK处理得到25/150个酶切类型,库容值达到92.00%。NM处理得到29/150个酶切类型,库容值达到92.31%。(2).多样性指数数值为CK<NM,且CK与NM处理amoA氨氧化古菌和amoA氨氧化细菌文库中,氨氧化古菌的Shannon-Wiener指数、丰富度指数、均匀度指数均低于氨氧化细菌。(3)氨氧化古菌的系统发育分析表明,所获得的氨氧化古菌均属于Crenarchaeota(泉古菌门),说明了泉古菌是黄土旱塬的优势菌群。本试验中得到的amoA基因序列与已知的来自土壤和沉积物中获得序列更相似,同源性更高。更多还原

【Abstract】 The loess plateau was an ancient rain-fed agricultural area in China, and the cultivated land area was 18 million hectares which account for 19% in China, so the further study of the loess plateau was significant. Due to erosion and drought, food production in the loess plateau was generally less than 2.0 t/hm2, which was lower than the national level. Although using chemical fertilizer was a necessary measure to increase crop yields, it could cause a large number of nitrate zone of invalid accumulation in the soil. Studies showed that there was a clear phenomenon of nitrate accumulation in soil by using a large number of nitrogen fertilizer in loess area. It not only caused high level of nitrogen waste and soil nitrogen fertility reduction, but also leads to the pollution for groundwater. Ammonia oxidation was the first reaction step of nitrification, rate-limiting step and the central link in the global nitrogen cycle. Therefore, the study of ammonia-oxidizing microorganisms (bacteria and archaea) had an important environmental significance. Study of soil ammonia-oxidizing bacteria and ammonia-oxidizing archaea community’s structure and diversity could provide the necessary theoretical basis to understand the characteristics of soil nitrification.The long-term field experiment was located in the Changwu State Key Experimental Station for Agriculture, Shannxi province, northwest China. The fertilization experiment included five treatments for each treatment in a randomized plot design. The five treatments were control without fertilizer (CK); those with combinations of fertilizer nitrogen, phosphorus and and organic manure:M, NM, PM, NPM. Fertilizers N, P and M were applied in the form of urea (120 kg N ha-1 per year), super phosphate (60 kg P ha-1 per year) and dairy manure (7500 kg ha-1 per year). Total DNA of five samples were extracted directly. Constructed amoA gene and Arch-amoA clone libraries, and the influence of CK, M, NM, PM and NPM fertilizer treatments on soil AOB diversity and community structure were analyzed by restriction fragment length polymorphism (PCR-RFLP). The main results were as follows:1. The soil ammonia-oxidizing bacteria amoA gene libraries were generated from the five treatments, the diversity and phylogenetic analysis showed that:(1) Total RFLP patterns of five treatments were 59(CK),56(M),83(NM),71(PM) and 42(NPM), respectively. The coverage (C value) of the clone libraries were 80.67%,80.67%, 60.00%,66.67% and 84.56%, respectively. Dominant ammonia-oxidizing bacteria existed in each treatment.(2) a-measurement indices analysis illuminated that there was differentia among five fertilizer treatments. The diversity was highest in NM treatment and lowest in NPM treatment. The Shannon-Wiener index and Species Evenness index represented an identical order NM>PM>CK>M>NPM, the Simpson index represented NM>CK>PM>NPM>M, and the Margalef index represented PM>NM>CK>M>NPM. The Sorenson index was 0.61 between M and NPM (M-NPM), which indicated there was a high community similarity. The lowest level of community similarity was existed in NM-CK and PM-CK, whose Sorenson indices were both 0.15. Sorenson indices of other treatments were from 0.23 to 0.38. The results indicated that long term fertilization resulted in change of AOB community.(3) Phylogenetic analysis of amoA gene amino acid sequences showed that Nitrosospira cluster 3 sequences was dominant in all treatments, but the proportion of clones in each treatment belonging to Nitrosospira cluster 3a or 3b was different, and some clones were affiliated with the Nitrosospira cluster 4. Different fertilizer environment could lead to the significant species variation of soil AOB.2. The soil ammonia-oxidizing archaea amoA gene libraries were generated from the five treatments, the diversity and phylogenetic analysis of ammonia-oxidizing archaea showed that:(1) Total RFLP patterns of CK treatment was 25, and the coverage (C value) of the clone library was 92.00%. Total RFLP patterns of NPM treatment was 29, and the coverage (C value) of the clone library was 92.31%.(2) a-measurement indices analysis illuminated that in terms of index value, CK<NM. Meanwhile, Shannon-Wiener index, Species Evenness index, Simpson index and Margalef index of ammonia-oxidizing archaea were lower than those of ammonia-oxidizing bacteria both in CK and NPM treatments.(3) Phylogenetic analysis of amoA gene sequences showed that all the sequences of the AOA belonged to an uncultured crenarchaeote, which was dominant in dry highland of loess plateau. The high community similarity and high homology existed in all the sequences we gained and the sequences gained from sediment and soil.更多还原