【作者】 刘岳燕；

【导师】 黄昌勇；

【作者基本信息】 浙江大学 ， 土壤学， 2009， 博士

【摘要】 水稻土对维持我国粮食生产和环境健康起着不可代替的作用。在粮食需求不断增加、水资源日益短缺、环境质量日趋恶化的压力下，维持水稻土壤持续高产性能、加强水资源合理利用、同时保证其生态环境健康，是我国当前迫切需要研究和解决的重要科学问题，也是当今全球关注的一个焦点。本文以我国典型稻田——太湖流域杭嘉湖平原的水稻土壤为研究对象，并运用近年来发展的土壤生物学新兴测试技术，如BIOLOG、PLFA、DGGE等，系统地研究了水分条件和水稻种植对土壤微生物学特性的影响，以及水分胁迫和水稻根际影响的土壤微生物活性和多样性随水稻生育期的演替特征，以期揭示水稻生育期内水分条件对水稻土壤质量的影响，获得如下主要成果：（1）研究了淹水土壤晾干预处理和不同保存温度对土壤微生物特性的影响。设计五种处理方式：淹水对照、淹水晾干、淹水冷冻、淹水晾干冷冻和淹水晾干冷藏处理。结合冷冻干燥技术，通过测定总磷脂脂肪酸含量和磷脂脂肪酸剖面，分析淹水土壤预处理和保存对土壤微生物生物量和多样性的影响。发现淹水晾干冷藏显著地减少土壤微生物生物量，而其它处理没有改变土壤微生物生物量。淹水土壤室内晾干预处理无论直接测定，还是在冷冻或冷藏保存下，其磷脂脂肪酸类群以及特征脂肪酸含量都发生了改变。通过进一步的对应分析，相对于其它处理，淹水冷冻处理的土壤微生物结构多样性与淹水对照处理更接近，表明了土壤直接冷冻后再冷冻干燥法对淹水土壤微生物结构多样性的影响较小。因此，我们提出直接冷冻干燥法是一种简单、方便预处理保存淹水土壤的方法，并且能保证土壤微生物群落结构变化最小。（2）针对传统使用的氯仿熏蒸浸提法在测定淹水土壤微生物生物量碳时出现的问题，以及在Innubushi，Witt等人改进后的方法基础上，进行了进一步的实验研究，提出采用液氯熏蒸提取-水浴法测定淹水土壤微生物生物量碳。明确了该方法在100℃水浴下排除剩余氯仿不会影响K₂SO₄浸提液中碳的损失，并确定水浴法排除液氯的时间（60min），液氯熏蒸用量(4-6μL g^-1干土)，并验证了该方法在淹水土壤中的熏蒸效率与常规氯仿熏蒸浸提法在旱地土壤中的熏蒸效率一致，而且熏蒸和非熏蒸重复间的重现性都较好，表明该方法应用于淹水土壤微生物生物量的测定是合适的。（3）采用BIOLOG碳素利用法、磷脂脂肪酸（PLFA）法和土壤酶活性测定等方法比较了三种水分条件（淹育、淹育晾干、非淹育）对水稻土微生物群落多样性及活性的影响。结果表明，淹育处理水稻土的脱氢酶、蔗糖酶活性明显高于淹育晾干和非淹育处理，并导致该土壤的基础呼吸升高。BIOLOG碳素利用法表明，非淹育处理的微生物群落平均吸光值（AWCD）显著低于淹育和淹育晾干处理。PLFA实验发现，淹育水稻土的真菌特征脂肪酸（18：2w6,9c）所占比例减少，真菌特征脂肪酸与细菌特征脂肪酸（15：0i+15：0a+16：0i+16：1w5c+17：0i+17：0a+17：0cy+17：0+18：1w7c+19：0cy）的比值下降；BIOLOG碳素利用法的群落水平生理剖面（CLPP）和PLFA测定结果经聚类分析后，发现淹育和淹育晾干处理的土壤微生物多样性在较低的距离尺度可聚成一类，且与非淹育土壤具有明显差异。淹育水稻土与淹育晾干相比，尽管土壤微生物群落结构和功能多样性有一定的相似性，但微生物的种群组成和活性仍发生了较大的变化。（4）在温室水稻种植条件下，根据水稻不同生育期的水分需求，研究干湿交替、水稻种植及其交互作用对土壤微生物学特性的影响。结果表明干湿交替使土壤基础呼吸速率和脱氢酶的活性下降，水稻种植减少了土壤脱氢酶的活性，交互作用使土壤基础呼吸速率、微生物代谢商和脱氢酶的活性下降。干湿交替、水稻种植及其交互作用显著增加了水稻移栽105天时好氧细菌、革兰氏阴性菌、革兰氏阳性菌的生物量。另外，干湿交替和交互作用使水稻移栽后25天和45天的细菌、放线菌、好氧细菌、革兰氏阴性菌、革兰氏阳性菌的生物量显著增加。干湿交替、水稻种植及其交互作用导致嗜甲烷菌（Ⅰ）在整个生育期内的含量远远高于长期淹育的对照处理。微生物结构多样性的聚类分析可清晰的看出，土壤微生物群落结构在水稻生长的前期主要受干湿交替和水稻种植的影响。但是在水稻生长末期，干湿交替对土壤微生物群落结构多样性的影响小于水稻种植。这一结果对水稻土壤的水分管理有一定的参考价值。（5）采用PCR-DGGE分子生态学方法研究了细菌和氨氧化菌微生物的遗传多样性。发现干湿交替，水稻种植以及交互作用明显改变了土壤细菌的遗传多样性，同样它们改变了氨氧化细菌和古菌的遗传多样性，相对来说，对氨氧化古菌的遗传多样性影响较小。长期淹水条件下的氨氧化古菌数量较少，这可能与其生长受到抑制有关。水稻土壤微生物遗传多样性的改变可能与干湿交替和水稻种植导致土壤含水量、含氧量、二氧化碳含量、养分状况等土壤物理、化学变化有关。更多还原

【Abstract】 Paddy soils play an important role in food production and environmental quality. With the increasing population pressure and decreasing water availability for agriculture, great attention has been paid to improving irrigation water management of paddy soils. Due to permanent or periodic prolonged saturation, the physical, chemical and microbial properties of paddy soils are different from wetland soils and aerobic soils. However, the measurements of soil properties are mainly originated from aerobic soils. In present study, a series of laboratory and pot experiments were conducted to investigate microbial biomass, basal respiration, enzyme activities, and microbial community diversity in relation to soil moisture regime and paddy growth stage. The results were summarized as follows:（1） The effects of pretreatment and storage of flooded soil on microbial characteristics were investigated. Five treatments including flooded （contrast）, flooded-air-drying, flooded-freezing, flooded-air-drying-freezing, flooded-air-drying-refrigeration were designed with pretreatment and different storage methods. Flooded and flooded-air-drying-freezing treatments were followed by freeze-drying before analyzing the total Phospholipid fatty acid （PLFA） and PLFA profile. The results showed that flooded-air-drying-refrigeration significantly decreased total PLFA, while other treatments were independent of air-drying or freezing. The contents of some PLFA groups and biomarkers were changed in response to air-drying pretreatment and different storage methods. Statistical analysis with correspondence analysis showed that air-drying and storage methods shifted the microbial community structure, but the effect of air-drying pretreatment followed by freezing and refrigenration on soil microbial community structure was more pronounced than direct flooded-freezing. These results indicated that deep freezing followed by freeze-drying may be the most recommendable procedure before soil biochemical analysis in flooded paddy soils.（2） Based chloroform-fumigation method, a chloroform-fumigaiton extraction-water bath method was developed for measuring microbial biomass carbon in flooded soils. Liquid chloroform was directly added to the flooded soil to be tested, which was fumigated for 24h at room temperature under normal atmospheric pressure and in darknesss, and then microbial biomass was extracted with 0.5 mol L^-1 K₂SO₄ solution. At the same time, non-fumigation control was extracted directly with 0.5 mol L^-1 K₂SO₄ solution. After a series of analyses with water bath time, chloroform concentration, and soluble carbon, the results showed that water bath under 100℃for 45-60 min with extraction did not affect content of soluble carbon, and this condition was enough to remove chloroform residue before measuring TOC. The developed method was tested on seven paddy soils which were incubated aerobically or anarobically and compared with the standard chloroform fumigation extraction. The results of the tests were reliable and reproducible, suggesting that chloroform-fumigaiton extraction-water bath method is a rapid and effective method for measuring microbial biomass carbon in flooded soil.（3） Influence of soil moisture regime （non-flooding, flooding-drying, and flooding） on microbial community diversity and activity was investigated by determining Biolog sole carbon source utilization pattern, Phospholipid fatty acid （PLFA） profiles and enzyme activity indices. Increased dehydrogenase and invertase activities were observed in Treatment Flooding as compared to what in the other two treatments, which apparently led to enhanced soil basal respiration. The average well colour development （AWCD） of the carbon sources on Biolog plates was significantly lower in Treatment Non-flooding than in the other two treatments. The fungal indicator （18:2w6,9c） and the proportion of fungal biomarker to bacterial biomarkers （15:0i+15:0a+16:0i+16:1w5c+17:0i+17:0a+17:0cy+17:0+18:1w7c+19:0cy） were lower under flooding conditions. Cluster analyses of the sole carbon source utilization and PLFA data demonstrated that Treatment Non-flooding differed from the other two treatments in soil microbial community. Although certain similarity was found between Treatment Flooding and Treatment Flooding-drying in structure and functional diversity of soil microbial community, the findings of the study suggested that there were some drastic changes in microbial community composition and activity associated with variation of soil moisture regime.（4） The effects of flooded-air-drying, paddy cropping, and their interaction on soil microbial properties were investigated. Flooded-air-drying decreased soil basal respiration and dehydrogenase activity, paddy cropping decreased soil dehydrogenase activity, and their interaction decreased soil basal respiration, metabolic quotient, dehydrogenase activity. Flooded-air-drying, paddy cropping and their interaction significantly increased the contents of aerobic bacterial, G-, G+ indicated by Phospholipid fatty acid （PLFA） in 105 days after transplating. Flooded-air-drying and interaction increased the contents of bacterial, actinomycetes, aerobic bacterial, G-, and G+. The content of type I methanotrophs indicator was significantly higher than control in the whole paddy growth stage. The cluster analysis of PLFA data showed that flooded-air-drying had lower effect on soil microbial community than paddy cropping.（5） Soil microbial genetic diversity of bacteria, ammonia-oxidizing archaea （AOA） and bacteria （AOB） were investigated using polymerase chain reaction-denaturing gradient gel electroporesis （PCR-DGGE）. Flooded-air-drying, paddy cropping, and their interaction treatment had significant effect on soil bacterial genetic diversity. The genetic diversities of AOA and AOB were also changed by the above treatments. However, the shift of microbial diversity was more variable in AOB than that in AOA. The content of AOA was more abundance in flooded-air-drying, paddy cropping and their interaction than that in control. The changes in bacterial, AOA and AOB genetic diversities may be due to the differences in soil water, oxygen, carbon dioxide, and nutrient contents induced by irrigation and paddy cropping.更多还原