【作者】 辜运富；

【导师】 张小平； 涂仕华；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 为跟踪和监测长期定位施肥对石灰性紫色土土壤质量的影响,四川省农业科学院于1982年在四川遂宁市船山区的石灰性紫色土上建立了“NPK长期肥料定位试验”,并对长期定位施肥下土壤的理化性质以及作物产量进行了长期研究。为深入认识长期定位施肥对石灰性紫色土质量的影响,本文在前人已有研究基础上,运用平板菌落计数和最大或然法（MPN）对不同施肥制度下的土壤可培养微生物数量进行了测定;利用化学分析法对土壤微生物量碳和氮、土壤呼吸作用、硝化作用以及土壤酶活进行了分析;最后,应用DGGE分子标记技术对长期不同施肥下细菌、AM真菌、古菌、硝化细菌和氨氧化细菌等微生物群落结构特征进行了研究。结果总结如下:（1）长期定位施肥后土壤微生物学特性和土壤活性的研究结果表明:与对照无肥处理（CK）相比,施肥能增加微生物数量,SMBC和SMBN的含量变化分别是10.8-91.4mg.kg^-1和10.8-37.2 mg.kg^-1,8种施肥方式中,NPKM处理下的土壤微生物量碳和氮最高。不同施肥方式对土壤中的酶活性具有不同影响,施肥能够增加土壤中转化酶、脲酶和磷酸酶的活性,而降低多酚氧化酶的活性。同样施肥也可以提高土壤呼吸作用强度,8种施肥处理中,CK处理的土壤呼吸作用最弱,NM肥料处理的土壤呼吸作用最强。施肥对土壤硝化作用表现为正向促进作用,与长期单施化肥相比,长期化肥配施农家肥对土壤硝化作用的促进作用最明显,不同作物种植方式也会影响土壤硝化作用,表现为紫色水稻土旱季硝化作用大于淹水土壤。（2） DGGE图谱分析表明不同施肥制度处理下土壤微生物群落结构出现了变化。N、P、K等无机肥料配合施用以及无机肥料与农家肥配合施用能够增加土壤细菌群落复杂性,特别是农家肥的处理,能够增加土壤中一些特殊的细菌类群,8种施肥处理中,在NPKM（或NM）处理的土壤细菌多样性最高,CK处理的多样性最低。DGGE条带序列分析表明不同施肥处理下土壤的优势共同条带与科克斯体科的lusitana菌（Aquicellalusitana）及酸杆菌纲（Acidobacteria）的细菌非常相似。（3）对长期不同施肥处理下的AM真菌群落结构特点的研究结果表明,石灰性紫色土上种植小麦的AM真菌多样性指数较种植水稻的大;8种施肥处理中,NM肥处理下的AM真菌多样性指数最高,农家肥配施无机肥的处理会提高AM真菌的多样性指数,施磷肥处理会降低AM真菌的遗传多样性:聚类分析表明土壤在种植水稻后,不同施肥处理的AM真菌的聚类分析表明,供试8种施肥处理土壤样品在0.53的水平上共分为四个群,N、CK、NM、NPKM和M为一个群;NP、NPK和NPM分别单独聚成另外三个群。在种植小麦后的聚类分析显示8种施肥处理土壤被分为三个族群:N、NM、NP、NPKM、NPM和M为一个群,NPK为第二族群,CK（无肥）独立为第三族群。（4）对长期不同施肥处理下的古菌群落结构特点的研究结果表明,长期定位施肥对土壤中的古菌形成明显的影响,具有不同的DGGE图谱。NP,NM和NPKM长期肥料处理下的土壤古菌多样性指数低于M、NPM、CK、N和NPK肥料处理。在DGGE图谱的基础上,分别选择种植水稻和小麦的NPK处理土壤DNA为样品,对土壤的古菌进行16S rDNA克隆测序分析,结果显示石灰性紫色土中的土壤古菌属于泉古菌界的陆生族里。系统发育分析发现,所有古菌克隆子聚在两个群里,群Ⅰ的古菌与前人分离自淡水池里的古菌非常相似。群Ⅱ的古菌与大陆土壤里的古菌非常相似。对DGGE图谱的聚类分析发现,不管是石灰性紫色土种植水稻还是小麦,8种施肥处理都聚在3个群里。种植水稻时,M和NPM肥料处理下的土壤古菌聚成第一个群,NP处理下的聚成第二个群,另外5种施肥处理（包括NPKM,NM,CK,N和NPM）聚成第三个群。种植小麦时,NPKM和M处理下的土壤古菌聚成一个群,NP处理下的聚成第二个群,N,NPK,NM,NPM和CK处理下的聚成第三个群。显示不同作物种植对土壤古菌也有一定影响。（5）利用DGGE技术研究了不同施肥制度对氨氧化细菌群落结构的影响。与对照无肥处理相比,施肥能改变氨氧化细菌的群落结构。无机肥配施农家肥的氨氧化细菌的群落结构丰富度比施用无机肥的处理高。主成分分析将8种施肥处理划分成两个主成分。植稻土壤,主成分1为NP、NM、NPM和NPKM,主成分2为CK、N、M和NPK;植麦土壤,主成分1为M、NM、NPM和NPKM,主成分2为CK、N、NP和NPK。主成分1的氨氧化细菌群落结构丰富度高于主成分2。水稻收获后土壤的氨氧化细菌群落结构丰富度高于小麦收获后土壤。（6）采用DGGE技术研究了长期不同施肥制度对石灰性紫色水稻土硝化细菌群落结构的影响。结果表明,施用化肥以及化肥配施有机肥会改变土壤中硝化细菌的群落结构。与长期单施化肥相比,长期化肥配施农家肥会提高土壤硝化细菌群落结构多样性。该紫色水稻土在种植水稻后的聚类分析（UPGMA）表明,供试8种土壤样品共分为三大族群:NP单独为一种族群,NPK、M、NM、NPM和NPKM为一个族群,CK,N为第三族群。在种植小麦后的聚类分析（UPGMA）显示参试8种土壤也被分为三个族群:CK、M、NM、NPM和NPKM为一个族群,NP与NPK为第二个族群,N独立为第三族群。更多还原

【Abstract】 In order to address and monitor the effect of long-term fertilization on the soil quality, the Soil and Fertilizer Institute of Sichuan Academy of Agricultural Sciences established a ’N, P, K long-term fertilization field experiment’on the Calcareous Purplish Soil in 1982 located in Chuanshan district, Suining city, Sichuan province, China. The impact of the long term fertilization on soil physiochemical properties and crop yields were studied extensively. Based on the previous works, the pour plate count method and most probable number method were used to study the impact of different long term fertilizers on soil microbes’ quantity. Chemical analysis was employed to determine the influence of long-term application of fertilizers on soil microbial biomass carbon and nitrogen, soil respiration, nitrification rates and enzymes activity. And finally, the Denaturing gradient gel electrophoresis （DGGE） molecule fingerprint method was applied to study the community structure of bacteria, AM fungi, archaea, ammonium oxidizing bacterial and nitrobacteria. The results were listed as following.（1） The results of long-term application of different fertilizers on soil microbial characteristics and activity in calcareous purple paddy soil showed that the treatments receiving any fertilizer application tended to increase the number of soil microbes compared with CK （no fertilizer treatment）. The contents of SMBC and SMBN were among 10.8-91.4 mg.kg^-1 and 10.8-37.2 mg.kg^-1, respectively. Among the 8 fertilizer treatments, the highest amounts of SMBC and SMBN were the soil amended with NPKM. Different fertilizer treatments made varied impact on the soil enzyme activity. Fertilization could improve the activities of invertases, urease and phosphatase, but decrease the activities of polyphenoloxidase. Soil respiration was also improved with fertilization. The respiration in the soil amended with NM was the highest while it in the soil with CK was the lowest. The soil nitrification rates of the same soil varied substantially after long-term treatments with various fertilizers. Generally, soil amended with any fertilizers tended to increase nitrification rates compared with the control. The nitrification rates of the soil appeared to be lower under rice cultivation than those of the same soil under wheat cultivation.（2） Soil bacterial community structure was analyzed by PCR-DGGE targeting bacterial 16S rRNA genes. The results showed that higher diversity of the soil bacterial community was found in soil amended with farmyard manure plus mineral fertilizer than in other fertilizer treatments. Some specific band emerged in the soil amended with farmyard manure. The highest diversity of bacterial communities was found in the NPKM treated soil. The bacterial community structures differed in rice and wheat plots. Sequencing of PCR products separated in DGGE showed that some of the common and dominant bands were closely related to Aquicella lusitana and Acidobacteria.（3） The results about the impact of long-term fertilization on the AM fungi community showed that the diversity of AM fungi was affected by different fertilization treatments and different crop cultivation. The Shannon diversity index of the soil AM fungi community under different long term fertilization with wheat cultivation was higher than that in the soil with rice cultivation. Among the eight long term fertilizer treatments, the highest Shannon diversity index appeared in the soil amended with NM, the diversity index of AM fungi was improved as the soil exposed to the fertilizer treatments with manure （M, NM, NPM and NPK）, while the application of phosphor fertilizer would decrease the diversity index. Cluster analysis showed that when the coefficient index was 0.53, in the soil after rice culitivation, the AM fungi communitiy in soil amended with N, CK, M, NM, NPKM was in the first cluster, fertilizers was in the first cluster. AM fungi communities exposed to the NP, NPK and NPM fell into the other three clusters, respectively. In the soil after wheat ciltivation, AM fungi communities in N, NP, M, NM, NPM and NPKM fertilizer treatments were clustered into one group, that in NPK to another group, and that in CK as the third cluster.（4） Archaea community structure in Calcareous Purplish Paddy soil under a long term fertilization experiment was studied. The results showed long term fertilizer could make great impact on the soil archaea community. Denaturing gradient gel electrophoresis （DGGE） analysis revealed that wheat and rice cropping had different impact on the soil archaea community structure, the richness and diversity of archaea community in the soil under NM, NP and NPKM were lower than those under the other fertilizer treatments （as M, NPM, CK, N, and NPK）. The sequences analysis of cloned 16S ribosomal DNA showed that the archaea in this study fell into two groups within the terrestrial cluster of Crenarchaeota. One group was closely related to clones from freshwater reservoir, while the second group was similar to clones found in terrestrial habitats. Cluster analysis of the DGGE profiles showed that archaea communities under 8 fertilizer treatments with rice and wheat cultivation were all clustered into 3 groups. In the soil with rice cultivation, the archaea communities in soil amended with M and NPM were in the same cluster. Archaea communities exposed to NP fell into the second group, while those exposed to the NPKM, NM, CK, N and NPM treatments were in the third group. In the soil with wheat cultivation, archaea community under NP was clustered into a single cluster. Those under NPKM and M were fallen into the second cluster, while those in N, NPK, NM, NPM and CK fertilizer treatments were in the third cluster.（5） Soil ammonium oxidizing bacterial community structure was analyzed by using DGGE technique. The treatments receiving any fertilizer application tended to alter the ammonium oxidizing bacterial community compared with the control. Among the eight fertilizer treatments, soil samples from the treatments of mineral fertilizers in combination with farmyard manure acquired more complex ammonium oxidizing bacterial community structure than those receiving mineral fertilizers alone. The principal component analyses （PCA） for ammonium oxidizing bacterial community structure showed that the eight fertilizer treatments were grouped into two PCAs. In the soil after rice harvested, PCA1 was including NP, NM, NPM and NPKM fertilizer treatments, PCA2 were consisted of CK, N, M and NPK fertilizer treatments. In the soil after wheat harvested, PCA1 was formed by M, NM, NPM and NPKM fertilizer treatments. PCA2 was composed of CK, N, NP and NPK fertilizer treatments. The richness of ammonium oxidizing bacterial community in PCA1 was higher than that in PCA2 and also higher in the soil after rice harvested than that after wheat harvested.（6） DGGE method was employed to determine the influence of long-term application of fertilizers on nitrobacteria community in calcareous purple paddy soil. The results showed that fertilizer amendment altered the nitrobacteria community structures. As compared to the mineral fertilizer treatments, the soil amended with the combination of the mineral fertilizers and farmyard manure increased the diversity of the nitrobacteria community as revealed by Unweighted Pair Group Method Clustering Analysis （UPGMA） of he DGGE banding patterns and cluster analysis of the DGGE profiles. In the soil after rice culitivation, the nitrobacteria communitiy in soil amended with nitrogen-phosphor （NP） fertilizers was in the first cluster. Nitrobacteria communities exposed to the fertilizer treatments with manure [manure only （M）, nitrogen plus manure （NM）, nitrogen and phosphor plus manure （NPM）, and nitrogen, phosphor and potassium （NPK）] fell into the second group while those exposed to the CK, N fertilzer treatments were in the third group. In the soil after wheat ciltivation, nitrobacteria communities in CK, M, NM, NPM and NPKM fertilizer treatments were clustered into one group, those in NP, NPK to another group, and those in N as a single cluster.更多还原