【作者】 罗红燕；

【导师】 谢德体； 蒋先军；

【作者基本信息】 西南大学 ， 农业资源利用， 2009， 博士

【摘要】 土壤中的微生物居住在一个由土壤颗粒控制的环境里,这些土壤颗粒的特性、形状和大小各异,并且具有高度复杂的空间分布与组成。微生物活动不仅对N的矿化,反硝化,生物固N,C、N循环等生化过程有较大影响,而且影响土壤结构的稳定性以及土壤中有机污染物的降解。土壤水稳定性团聚体是最重要的土壤结构体,了解微生物在土壤团聚体中的分布对于预测这些过程的发生以及反应速率是至关重要的。不同气候和土壤条件下的研究表明,大小不同的团聚体中有机碳含量、微生物数量及种群有较大差异。由于土壤微生物生物量对土壤环境因子的变化极为敏感,农业中的耕作方式、轮作措施、有机残体的投入与施肥量都可能引起土壤微生物生物量的改变。因此需要更多在不同气候和土壤类型下的研究。本文以亚热带地区长期定位试验紫色水稻土为研究材料,涉及3个处理:垄作免耕(RNT)、冬水稻田(FPF)和常规平作(CT),在全面了解长期保护性耕作下亚热带地区紫色水稻土基础上,运用常规分析技术和方法表征土壤团聚体组成及团聚体的孔隙和水分、营养元素特征,并采用麦角固醇和胞壁酸标记法测定各级团聚体中真菌和细菌的生物量,同时运用氯仿熏蒸浸提法、平板培养法、PCR-DGGE法分析三种不同耕作方式对团聚体中微生物分布的影响。主要结果如下:(1)土壤不同耕作制度对土壤团聚体及团聚体水分和孔隙的影响:＞2.0mm水稳定性团聚体的数量:垄作免耕＞冬水稻田＞常规平作;垄作免耕的土壤容重为1.18,小于冬水稻田的1.23和常规平作的1.28,土壤容重小,结构疏松,总孔度垄作免耕52.0%,大于冬水稻田的50.0%和常规平作的46.0%。在相同的土壤水势下,土壤的含水量相关系数r~2垄作免耕土壤为0.997,大于冬水稻田的0.903和常规平作的0.877,垄作免耕的含水量大于冬水稻田和常规平作土壤。(2)在同一耕作制度下,不同粒径的团聚体之间,0.053～0.25mm粒径的有机碳、全磷、全钾和全氮含量在各分布中最低(p＜0.05):＜0.25mm,团聚体直径愈小,交换性阳离子、有机碳、全磷、全钾和全氮含量愈高;团聚体粒径＞0.25mm,,随团聚体直径改变,有机碳、全磷、全钾和全氮含量没有明显规律性。与冬水稻田和常规平作处理相比,垄作免耕可显著增加各级团聚体的养分储量。(3)微生物生物量碳、氮及可溶性有机碳在亚热带地区紫色水稻土水稳定性团聚体中的分布主要受团聚体结构本身的影响(p＜0.05),而耕作措施对其分布模式的影响不显著(p＞0.05);微生物生物量碳在土壤团聚体中分布均匀,微生物生物量氮与可溶性有机碳在＜0.25mm微团聚体中含量最高。垄作免耕有利于提高土壤团聚体中的微生物生物量及可溶性有机碳含量。(4)采用麦角固醇和胞壁酸标记法测定各级团聚体中真菌和细菌的生物量。结果显示在垄作免耕、冬水稻田、常规平作土样中,真菌生物量与细菌生物量在＜0.053mm的粘粒中含量最低;在0.053～0.25mm的土壤微团聚体中最高;在＞0.25mm的大团聚体中真菌和细菌生物量随粒径的增大而增多,但细菌和真菌生物量在不同耕作方式的土样中的变化模式有一定的差异。垄作免耕的土壤真菌和细菌的生物量在三种耕作方式中最高,冬水稻田次之,常规平作最低。真菌生物量与细菌生物量的比率分布模式在不同耕作方式的土样中差异较大,垄作免耕的真菌生物量与细菌生物量的比率在三种耕作方式中最高,冬水稻田次之,常规平作最低,但除了在0.25mm～1.0mm、2.0mm～4.76mm的团聚体之间有明显差异外(p＜0.05),其余粒径之间均无明显差异(p＞0.05)。_十壤真菌生物量和细菌生物量在亚热带紫色水稻土水稳定性团聚体中的分布模式与团聚体的粒径和耕作方式有关,三种耕作方式中垄作免耕的影响最为明显。(5)＜0.25mm的土壤团聚体,随着粒径的减小,真菌数量增加;粒级为0.053～0.25mm的土壤团聚体,真菌数量最低(p＜0.05),垄作免耕、冬水稻田和常规平作分别为1.52、0.84、0.73(10~4个/g.干土)。＞0.25 mm,团聚体粒径越大,真菌的多样性指数改变没有明显规律性。＜0.25mm,团聚体粒径越小,真菌的多样性指数越高。真菌的丰富度垄作免耕＞冬水稻用＞常规平作。在垄作免耕土壤中存在大量的菌丝态真菌,表明免耕具有较稳态的土壤环境和较稳态的生物类群。(6)六种不同的DNA提取方法中,改良的DNA kit法效果最好。采用基因V3区具有特异性的引物对,对每个土壤样品中的微生物基因组进行特异性的扩增。均获得了长约230bp的产物。PCR产物经变性梯度凝胶电泳(DGGE)进行分离后得剑不同数目且分离效果较好的电泳条带。通过计算Shannon多样性指数,发现土壤细菌多样性在亚热带地区紫色水稻土水稳定性团聚体中的分布模式主要与耕作方式有关,团聚体的粒径大小有一定程度的影响。三种耕作方式细菌基因多样性变化规律为垄作免耕＞冬水稻田＞常规平作。因此,亚热带地区紫色水稻士的孔隙度及水分特征曲线受耕作方式的影响,垄作免耕可促进较大的孔隙度及含水量。团聚体中的营养元素分布受耕作方式和团聚体粒径大小的共同影响;不同粒径团聚体中微生物总生物量碳氮,细菌和真菌的生物量主要受团聚体粒径大小的影响,团聚体中各优势种属的多样性主要受耕作方式的影响。垄作免耕提高了细菌和真菌优势种属在不同大小团聚体中的丰富度。本文利用生物标记法和分子生物学方法从不同耕作制度和团聚体尺度研究亚热带地区紫色水稻土团聚体中细菌和真菌生物量及多样性的分布特征,探讨了与之相关的物理和化学营养元素机制,为从团聚体层面深入研究土壤细菌和真菌在土壤营养元素稳定、积累和转化过程中的重要作用,获得了相关基础信息。由于土壤营养元素在土壤团聚体中的发生与所在地块的土壤类型、地理位置、农耕措施、水文条件、土地利用、施肥管理等有关,目前农田土壤团聚体中的物理化学生物学变化的循环制约尚无统一的评价指标,开展相关研究,可对农田土壤施肥进行更精准的定量预测,为区域土壤肥力的宏观管理提供理论依据。更多还原

【Abstract】 The soil microorganisms living in a controlled environment by the soil particles, has a highly complex spatial distribution and composition. In the field of agriculture and the environment, microbial activity has significant impact on the biochemical processes, such as: N mineralization, denitrification, bio-solid N, C, N cycle, the stability of soil structure and degradation of organic pollutants in soil and so on. The stability of soil aggregates is the most important factor on soil structure. It is essential for researching the distribution of micro-organisms in the soil aggregates to understand and predict the occurrence of these processes as well as the reaction rate.Under different climatic and soil conditions, research has shown that the organic carbon content, microbial population and community are quite different in different sizes of aggregates. The microbial biomass are extremely sensitive to the changes of agriculture farming methods, such as crop rotation measures, organic debris input and fertilizer can cause changes in soil microbial biomass. Therefore, more study in different climatic and soil types is needed.In this paper, The composition characterize of soil aggregates and aggregate porosity and moisture, The distribution patterns of nutrient and bacteria and fungi, The diversities of bacteria and fungi in different water-stable aggregates (WSA), and the effects of tillage methods on the number of nutrient and bacterial and fungal biomass, were investigated in a long-term field experiment. Tillage treatments examined in this study included Conventional Tillage (CT), Flooded Paddy Field (FPF), Combining Ridge and No-tillage (RNT). A wet sieving method was used to separate soil aggregates into six groups based on particle diameter: >4.76 mm, 2.0～4.76 mm, 1.0～2.0 mm, 0.25～1.0 mm, 0.053～0.25 mm and <0.053 mm. The techniques and methods such as the conventional analytical, the chloroform fumigation extraction method, plate culture method, and PCR-DGGE Analysis were used. Main results are as follows:(1) The affection of different cropping systems on composition of soil aggregates and the distribution of soil moisture and porosity: The composition of water-stable aggregates about > 2.0mm, combining ridge and no tillage (RNT)>flooded and paddy field (FPF)>conventional tillage (CT); The porosity of RNT is more bigger than the FPF and CT, The structure and the distribution of pore of RNT is better than the FPF and CT, and soil bulk density of RNT is small and soil structure is loose with a total porosity large, well to keep the soil moisture, can promote the formation of soil aggregates and could be better coordination of water status. In the same soil water potential, the soil moisture content of combining ridge and no tillage (RNT) is greater and more effectiveness than flooded and paddy field (FPF) and conventional tillage (CT).(2) The nutrient content of top soil aggregate size: In the same farming system, different aggregates size, the organic carbon, total phosphorus, total potassium and nitrogen content in the size of 0.053～0.25mm is the lowest distribution (p<0.05); while in the diameter of <0.25 mm, the smaller aggregates size, the higher content of organic carbon, total phosphorus, total potassium and total nitrogen. No regularity of the content of total organic carbon, total phosphorus, total potassium and total nitrogen (p<0.05) in aggregate size of >0.25mm. And combining ridge and no tillage (RNT), can significantly increase the levels of nutrients reserves in aggregates.(3) Microbial biomass carbon, nitrogen distribution: microbial biomass carbon, nitrogen, and dissolved organic carbon in subtropical areas of purple paddy soil aggregate water stability in the distribution of mainly affected by the impact of aggregate structure itself (p<0.05), The effects of tillage was not significant (p> 0.05); microbial biomass carbon in soil aggregates distributed evenly, microbial biomass nitrogen and dissolved organic carbon in the <0.25mm micro-aggregates of the highest concentrations. Combining ridge and no tillage (RNT) will help to improve soil aggregates in the microbial biomass and soluble organic carbon content.(4) Fungal and bacterial biomass was estimated by measuring the amount of ergosterol and muramic acid, respectively, in the samples. Both bacterial and fungal biomass were lowest in the smallest soil particles (<0.053 mm) and highest in the 0.053～0.25 mm fraction. As the size of the soil micro-aggregate increased, the associated bacterial or fungal biomass also rose, however, there were notable response differences between the two taxonomic groups. Of the three tillage methods examined, maximum bacterial and fungal biomass was found in the Combining Ridge and No-tillage (RNT) treatment; minimum biomass occurred under Conventional Tillage (CT). There were significant (p<0.05) differences in fungal and bacterial biomass in the 0.25 mm～1.0 mm and 2.0 mm～4.76 mm particle-size groups among the three tillage methods; there were no significant differences(p>0.05) in the remaining size groups.(5) The diversity of Soil fungi and the advantages of the fungi in different tillage methods: In the diameter of >0.25mm, with the size of soil aggregates increases, No regularity change of fungi; <0.25mm, with the decreasing size of soil aggregates, fungi increase in the number; the size of 0.053～0.25 mm of soil aggregates, the lowest number of fungi (p<0.05). The distribution of fungi is characterized by in the diameter of <0.25 mm. the smaller the aggregate size, the higher the diversity index. The most richness of fungal diversity exist in the combining ridge and no tillage (RNT), there is a large number of fungal hyphae, indicating that the combining ridge and no tillage (RNT) can improve soil fungi in K-response biological, and show a more steady state of no-tillage with soil environment and a more steady state of biological taxa.,(6) Extract the microbial genome of rice soil using kits in different soil aggregate size, and us the genome as a template, using the majority of bacteria and archaea genes V3 area with specific primers, for each soil samples of the microbial genome-specific amplification. The product of about 230bp by denaturing gradient gel electrophoresis (PCR-DGGE) to separate and obtain a better separation effect of different amounts and electrophoretic bands by calculating the Shannon diversity index and found that soil bacteria diversity in the subtropical areas of purple paddy soil aggregate water stability in the distribution patterns of tillage mainly related to the particle size aggregates a certain extent. Bacterial genetic diversity of three kinds of tillage:combining ridge and no tillage (RNT)> flooded and paddy field (FPF) and conventional tillage (CT).Therefore, the porosity numbers of soil and water availability were best in combining ridge and no tillage (RNT). Aggregate size of <0.25mm, soil nutrients with the aggregate reduce the size of the reserves increase. Aggregates of different total microbial biomass C and N, bacterial and fungal biomass and the number of dominant species of the genus, diversity affected by farming methods and the particle size of the aggregates. The combining ridge and no tillage (RNT) increased dominant species of bacteria and fungi,Aggregates of different sizes are in the richness and the evenness of the distribution.In this paper, biomass and diversity of fungal and bacterial in different aggregate size and different tillage on paddy soil of subtropical areas were estimated by bio-labeling and molecular biological methods. This research provided some information for predicting the important role of soil bacteria and fungi in soil nutrient stability、accumulation and transformation processes. Because the occurrence of soil nutrient elements in soil aggregates was related to the location of land、soil type、geographic、tillage practice、the hydrological conditions、land use、fertilizer management, the interrelated research can provided more accurate quantitative prediction for tillage fertilizing, and provided the macro-management theory about the region’s soil fertility.更多还原