【作者】 李琼；

【导师】 姚槐应；

【作者基本信息】 浙江大学 ， 土壤学， 2014， 硕士

【摘要】 硫是水稻必需的营养元素之一,而硫酸盐还原菌(sulfate-reducing bacteria, SRB)是稻田土壤中硫酸盐还原过程中重要的功能菌群。水稻田是典型的生态环境,经常被用来研究土壤功能微生物的群落结构。研究水稻田微生物群落结构的组成以及可能发生的变化,对于明确水稻土生物地球化学循环过程与线路,比较稻田土壤系统可持续发展等问题均具有重要意义。本论文以广州冲蒌酸性硫酸盐水稻土为对象,通过土壤DNA的提取、多聚酶链式反应(PCR)、变性梯度电泳(DGGE)和测序等一系列分子生物学手段,深入研究水稻土改良后土壤中细菌、古菌和SRB种群群落结构的变化情况,探讨了水稻土中硫素生物地球化学循环过程。论文的主要内容与研究成果如下：1、水稻田土壤主要化学指标-pH、土壤有机碳、有效磷和硫酸盐含量随着土壤改良而增加。水稻土主要微生物生化指标微生物量碳,与对照相比也有显著的增加。添加5%的生物炭的处理土样的微生物量碳增多的程度高于5%的有机肥和1%的石灰处理。2、本研究设计6个处理,分别为：(1)对照(CK),荒土；(2)冲蒌水稻土(改良前);(3)冲蒌水稻土(改良)；(4)添加1%石灰;(5)添加5%有机肥；(6)添加5%生物炭。用以研究水稻土中细菌的群落结构对不同施肥处理的响应。水稻田细菌的群落多样性在添加改良剂下明显增加,细菌的群落结构发生相应的改变。对细菌的16S rDNA基因进行分析得出,不同改良处理下水稻土细菌群落结构有一定的变化,但是优势类群没有明显变化。测序分析得到优势菌种的分类集中为放线菌门(Actinobacteria),也含有少量的存在于厚壁菌门(Firmicutes)和变形菌门(Proteobacteria)。而且它们与放线菌属(Actinomyces)、未培养细菌(Uncultured bacterium)、硫单胞菌属(Thiomonas)、变形菌(Deltaproteobacteria)的同源性较好。3、通过模拟培养试验,研究了酸性硫酸盐水稻土中添加石灰、生物炭和有机肥后,硫酸盐还原过程及SRB的群落动态变化。基于DGGE的聚类分析(Cluster Analysis)表明,在水稻的不同改良处理,荒土与自然改良的非根际水稻土中SRB的群落结构均发生了明显的改变,但是,短期模拟改良的土壤与荒土中的SRB的群落结构无显著差异。系统发育分析表明SRB分属于分属于Desulfonema, Desulfomicrobium, Desulfovibrio, Thiococcus, Thiobacillus和Uncultured SRB共5个SRB类群,其中未能培养的SRB为优势类群。4、不同改良的酸性硫酸盐水稻土壤环境中,古菌群落结构的多样性非常丰富,但由于其多样性受土壤酸碱度、水分、养分等多种因子的影响,在不同改良的土壤环境中其多样性存在差异。古菌在酸性土壤环境中种类最丰富,多样性指数最高。本实验向荒土中添加生物炭(5%)、有机肥(5%)和石灰(1%)土壤改良剂进行实验,然后研究不同改良剂对水稻土壤中古菌生态的影响。结果表明：与未作处理的荒土相比,添加改良剂的土壤中的古菌多样性减少。对部分优势菌种进行切胶测序发现,优势菌种与热变形菌纲(Thermoprotei)、未培养细菌(Uncultured bacterium)、甲烷杆菌纲(Methanobacteria)的同源性较好。更多还原

【Abstract】 Sulfur is one of the most necessary nutrients for rice growth. Sulfate-reducing bacteria(SRB) are the main functional microbial group and play important roles in sulfate reduction in the rice paddy soils. Acid sulfate soils contain iron sulfide.When the soils are drained, exposed to air by a lowering of the water falde, the sulfides react with oxygen to form sulfuric acid. In this study, a series of uncultivated and cultivated (paddy field) acid sulfate soils were used to test.The potential responses of bacteria、archaea and SRB community structure soil amendments, using culture-independent molecular techniques such as soil DNA extraction, polymerase chain reaction (PCR), real-time PCR, denaturing gradient gel electrophoresis (DGGE) and sequencing. The major contents and findings of the main results are as follows:1. The basic chemical properties in the paddy soil, i.e., soil pH, organic C, available P and sulfate concentrations increased significantly with soil modification.Soil microbial biomass C showed a significantly increase compared with the control. Biochar (5%) had higher effect on microbial biomass C than manure (5%) or lime (1%)2. The lateral community structure was investigated in order to understand the response to different amendments in rice paddy, including lime (1%), Biochar (5%), manure (5%). Significant differences in the bacterial were observed among different treatments, but the dominant taxa had no Significant differences.Sequence-based analysis of16S rDNA indicated that the dominant taxa were Actinomyces, Uncultured bacterium, Thiomonas and Deltaproteobacteria.3. The SRB community structure and sulfate reduction process in paddy soils amending with biochar manure and lime were examined in the culture experiment. No significant differences of SRB community structure were observed among different paddy soils amending with biochar manure and lime treatments, however significantly increased in the natural amended paddy soil. Phylogenetic analysis indicated that Desulfonema, Desulfomicrobium, Desulfovibrio, Thiococcus, Thiobacillus and Uncultured SRB were found, uncultured SRB were also found with a high proportion in the paddy soils.4. The archaea types were very abundance in different amended paddy soil. There were significant discrepancy in archaea diversity among different soil, due to the influence of pH, soil moisture, the content of carbon and other factors. High archaea biological diversity was found in the acidic soil. The results showed that there were some degree of decline in archaea community structure under the different amendments. The phylogenetic analysis based on the DGGE profiles showed that the archaea dominant taxa was Thermoprotei、Methanobacteria and Uncultured bacterium.更多还原