【作者】 靳振江；

【导师】 潘根兴；

【作者基本信息】 南京农业大学 ， 生态学， 2013， 博士

【摘要】 水稻土固碳是当前我国农业应对全球温室气体减排的需求驱动下的前沿问题,其中所涉及的土壤-作物-微生物之间的相互作用可能是水稻土碳循环的特异性问题。因此,研究水稻土微生物群落结构与功能变化规律有利于充实和发展土壤碳循环理论。水稻土固碳能力比旱地农田土壤的潜力更大,而耕作和和施肥在很大程度上影响和调控着水稻土的固碳强度。然而,对土壤-作物-微生物相互作用过程及其在耕作和不同施肥条件下的变化还缺乏深入认识。因此,本论文主要围绕稻田耕作后有机碳变化过程中微生物群落区系和活性变化特点,以深入认识水稻耕作栽培对土壤微生物群落结构和功能的影响；围绕不同施肥对成熟水稻土功能微生物群落结构和活性的影响,以认识稻田土壤微生物群落及活性的短期变化,从而为了解稻田土壤微生物变化特征及其对生物地学过程的可能影响积累科学资料。以湖北荆江地区起源于河流湿地的水稻土为研究对象,采集代表性河流湿地和稻田耕层土壤样本,用氯仿熏蒸-硫酸钾提取法测定微生物生物量碳,用稀释平板菌落计数法和聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)研究微生物区系数量和群落结构多样性,并配合比色法测定土壤酶活性。以设在我国江苏太湖流域长达23年的肥料定位试验田的水稻土为研究对象,采用传统的平板菌落计数法与PCR-DGGE结合基因克隆和测序等方法,以amoA、Arch-amoA、nirK、nifH和cbbLR分别作为指示基因,研究了氨氧化细菌(AOB)、氨氧化古菌(AOA)、反硝化细菌(NRB)、固氮细菌和CO2固定细菌等5类微生物在无肥处理(NF)、NPK化肥处理(CF)、化肥配施猪粪(CFM)和化肥结合秸秆还田(CFS)4种不同施肥处理下群落结构的变化,并采用归一化处理土壤酶活性和土壤养分强度,分析探讨土壤有机质-微生物-功能活性的关系变化,主要研究结果和认识如下：1.湿地土壤耕垦为水田对土壤微生物群落及活性的短期变化稻田土壤有机碳含量比湿地提高了55.42%,微生物生物量碳提高了180%。细菌、真菌、放线菌和自生固氮菌的丰度与细菌和真菌多样性均未发生分异,但是稻田土壤蔗糖酶、脲酶和碱性磷酸酶活性比湿地分别提高了89%、70%和72%。微生物生物量碳和归一化的酶活性均与土壤有机碳呈显著正相关关系。因此,经过近30年的耕种,湿地起源水稻土有机碳含量显著提高,微生物生物量碳与酶活性也显著增强。表明起源于湿地的水稻土在发育过程中,随着有机质积累,微生物丰度和生物化学活性快速提高,从而加快养分周转从而能促进水稻土功能演进。2.长期不同施肥下稻田土壤氮循环功能群微生物群落结构的变化长期不同施肥处理显著改变了土壤固氮细菌的群落结构。与NF处理相比,CF处理、CFM处理和CFS处理均显著提高了自生固碳菌的数量,提高幅度分别为0.58倍、0.66倍和1.06倍,而且,三个施肥处理下nifH基因丰度的提高幅度分别为2.13倍、10.79倍和3.44倍。自生固氮细菌的数量与SOC含量之间具有显著的正相关(R2=0.9378,P=0.0319),nifH基因丰度与归一化的土壤养分呈显著正相关(R2=0.9707,P=0.0147)。与NF处理相比,CF处理和CFM处理下土壤微生物固氮酶活性均提高了2倍。但是,长期施肥下土壤固氮酶活性与可培养固氮细菌数量和nifH基因丰度之间均没有相关性。长期不同施肥处理显著改变了土壤氨氧化细菌、氨氧化古菌与反硝化细菌的群落结构。与NF处理相比,CF处理、CFM处理和CFS处理下硝化细菌amoA基因丰度的提高幅度分别为1.36倍、39.57倍和29.98倍,CFM处理和CFS处理下氨氧化古菌Arch-amoA基因丰度的提高幅度分别为446.97倍和88.19倍,反硝化细菌nirK基因丰度的提高幅度分别为0.51倍、2.81倍和0.80倍。不同施肥处理下的amoA基因的丰度与Arch-amoA基因丰度的变化趋势相同,但是后者的丰度值较前者低2-4个数量级。amoA基因丰度和nirK基因丰度均与归一化的土壤养分呈显著正相关(R2=0.9101,P=0.0460；R2=0.9910,P=0.0006),表明施用有机肥处理下土壤有机质积累较多与养分平衡有利于提高氨氧化细菌/古菌与反硝化细菌的丰度。土壤有机质(SOC)与硝化势之间具有显著的正相关关系(R2=0.9110,P=0.0470); CFM处理下有较低的反硝化势,而CFS处理下的反硝化势较高,表明猪粪有机肥处理有利于N2O减排。不过,不同施肥处理下的amoA/Arch-amoA基因丰度与土壤硝化势之间没有相关性,nirK基因丰度与土壤反硝化势之间也没有相关性。3.长期不同施肥对稻田土壤碳循环功能菌——CO2固定细菌群落结构变化长期不同施肥处理显著改变了土壤的丰度。与NF处理相比,CF处理、CFM处理和CFS处理下cbbLR基因丰度的提高幅度分别为2.13倍、10.79倍和3.42倍,cbbLR基因丰度与归一化的土壤养分呈显著正相关(R2=0.9707,P=0.0147)。综上所述,无论是湿地开垦为稻田后的微生物生物量及生物化学活性变化,还是长期施肥下成熟水稻土氮循环功能群丰度及多样性变化,均响应于土壤有机碳和养分条件的变化。无论是起源于湿地的水稻土土壤微生物区系总体生物量和酶活性,还是氮循环功能群的丰度和多样性,均与有机质含量变化具有正相关关系,且随着其含量提高,基因丰度和多样性大幅度提高,因此稻田有机质积累改变了微生物生存条件,有机质-微生物-酶活性关系呈演进性增强,从而促进土壤生物功能和农田生产力,有利于稻田生态系统的增强和稳定。另外,不同施肥处理下功能群微生物基因丰度与氮素转化的活性之间并没有观察到相应变化,说明有机质积累下微生物丰度的提高,增强了生态系统功能群微生物的冗余度,这可能预示生态系统功能的稳定性的内在机制,但尚需更多试验验证。本研究还表明,有机无机肥配施能够提高稻田土壤微生物的硝化活性和固氮活性,降低反硝化活性。更多还原

【Abstract】 At present, carbon sequestration in a paddy soil is a front-line issue under the background of globle climate changing and of demanding for decreasing green-house-gas emission in our country’s agriculture. The interaction among soil, rice and microbe involved in the process of carbon sequestration is maybe a special issue of carbon cycle in paddy soil. So, the study of microbial community structures and functional activities is needed which can encrich and develop the theory of soil carbon cycle. Both of rice cultivation and different fertilizations can largely affect the intensity of carbon sequestration in rice paddy where there was a higher potential of carbon sequestration than that in dryland. In this paper, two subjects are invesgated for understanding the changing of the interaction among soil, rice and microbe under rice cultivation and different fertilizations. One is what are changes of microbial floras and activities with soil organic carbon (SOC) in rice paddy cultivated from natural wetland. The other is what are changes of functional microbes under different fertilizations in a mature rice paddy. Probing the two problems can help us to understand the changing law of microbes in a paddy soil and can provide the scientific data for the law’s application in bio-geo-process.In the first study, the topsoil samples were collected from natural wetlands and rice paddies in Jingjiang region, Hunbei, China. SOC and microbial biomass carbon (SMB-C) were examined with sulfate digestion method with potassium dichromate and chloroform fumigation combining extraction with lemery, respectely. Microbial flora numbers and community structure diversities were measured with plate counting method and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), respectely. Microbial activity was determined with soil enzyme assays as well. In the second study, the topsoil samples were collected from a23-year fertilizing paddy field in the Tai Lake region, Jiangsu, China where the fertilization treatments were designed as follows:no fertilizer application (NF), chemical fertilizers only (CF), chemical fertilizers plus pig manure (CFM), and chemical fertilizers plus rice straw return (CFS). The methods of plating and PCR-DGGE combining gene cloning and sequencing were adopted for investing the soil microbial communities of nitrifying, denitrifying, azotobacter and CO2-fixing under four treatments with amoA and Arch-amoA, nirK, nifH and cbbLR gene as the indicators of ammonia oxidizing bacteria (AOB) and archaeal (AOA), denitrifing bacteria (NRB) and azotobacter and CO2-fixing bacteria, respectively. Normalized method was using for charactering overall soil enzyme activities and soil nutrients richness, which is help for discussion the relativity changes of SOC, microbe and functional activity. The main results were as follows:1. Changes of soil microbial community and activity in a short time in a rice paddy cultivated from natural wetland.The SOC content increased by55.42%in rice soils compared to wetland. SMB-C was found as180%higher in paddy soil than in wetlands. There was no significant difference in abundance of the bacterial, fungi, actionmycetes and autotrophic azotobacter, and diversity of both bacterial and fungi between wetlands and paddy field. However, the observed microbial enzyme activities of invertase, urease and alkaline phosphatase enhanced by89%,70%and72%, respectively, in paddy field over wetlands. Statistical analysis revealed a significant correlation of SMB-C and normalized overall enzyme activity with soil organic carbon contents, respectively. These observations supported that the size of soil microbial biomass and microbial functional activity with enzymes were enhanced as SOC storage increased due to rice cultivation for several decades, which indicated that microbes promote SOC accumulation through accelerating nutrients turnover in a paddy’ development.2. Changes of microbe communities involved in N-cycle in a rice paddy under long-term fertilization treatmentsThe long-term fertilization treatments had changed community structure of Azotobacter. The clony forming unite (CFU) of Azotobacter under CF treatment, CFM treatment and CFS treatment were found as higher0.58,0.66and1.06times, respectively compared with that in NF treatment, the abundances of nifH as well as were found as higher2.13,10.79and3.44times. Statistical analysis revealed there was a significant correlation of the CFU of Azotobacter with SOC (R2=0.9378, P=0.0319), as well as the abundance of nifH with SNRN of SOC, T-N and T-P (R2=0.9707, P=0.0147). The soil nitrogen fixation activities under CFM and CFS treatments were found as higher204%and196%, respectively compared with that in NF treatment. However, there was not a significant correlation of the soil nitrogen fixation activity with the CFU of Azotobacter or the abundance of nifH gene.The long-term fertilization treatments had changed community structures of AOB AOA and denitrifier significantly. Compared with the microbial abundance in NF treatment, the abundances of amoA and nirK gene in CF, CFM and CFS treatment were as higher1.36,39.57and29.98times and0.51,2.81and0.80times, respectively, as well the abundances of Arch-amoA gene under CFM and CFS treatments were as higher446.97and88.19times, respectively. The abundance of amoA is two to four orders higher than the one of Arch-amoA though both are the same change trend across the four treatments. There were significant correlations of abundance of amoA and nirK with soil nutrient richness normalized (SNRN) of soil organic carbon (SOC) and total nitrogen (T-N)(R2=0.9101, P=0.0460) and of SOC, T-N and total phosphorus (T-P)(R2=0.9101, P=0.0460), respetively, which indicated that SOC accululation and nutrient richness under manure treatments can increase the abundances of AOB, AOA and denitrifier. There was higher soil nitrification potential under CFS treatment and a positive correlation between SOC and soil nitrification potential (R2=0.9110, P=0.0470). There was a highest denitrification potential in CFM treatment while a lowest one in CFS treatment, which indicated a large reducing potential of N2O emission under pig manure treatment. However, the abundances of amoA/Arch-amoA and nirK were decoupling with the soil nitrification potential and denitrification potential across four treatments, respectively.3. Changes of functional microbe involved in carbon cycle——CO2-fixing bacteria community in a paddy soil under long-term fertilization treatmentsThe long-term fertilization treatments had changed community structure of microbe involved in carbon cycle. The abundances of cbbLR under CF treatment, CFM treatment and CFS treatment were found as higher2.13,10.79and3.42times, respectively compared with that in NF treatment. Statistical analysis revealed there was a significant correlation of the abundance of cbbLR with SNRN of SOC, T-N and T-P (R2=0.9920, P=0.0020).In summary, not only the changes of both soil microbial biomass and bio-chemical activities in a rice paddy reclaimed from wetland, but also th changes of both the abundances and structures of microbe involved carbon and nitrogen cycle under long-term fertilization response the changes of SOC and nutrients. Moreover, there was positive relationship between the above microbial charisteres and SOC content, which showed that there was a substantially increaseing of abundance and diversity along with SOC increasing. The above results indicated the SOC accumulation changed the living condition of microbe so that the relationship among SOC, microbe and enzyme activity was evolutionarily buding up, which enhanced soil bio-function and crop production and favored the rice paddy ecosystem stable. In addition, significantly coorelation between functional microbial abundances and activities involved in N transform under different fertilization treatments were not observed, which indicated that SOC accumulation increased the redundancy rate of functional genes. This is, therefore, probably inner mechanism of the stability of rice ecosystem which deserved the further study. In this study, the result showed that a decreasing denitrification activity but increasing avtivities of nitrification and nitrogen fixation under the CFM treatment in a rice paddy as well.更多还原