【作者】 王军；

【导师】 朱鲁生；

【作者基本信息】 山东农业大学 ， 农药学， 2012， 博士

【摘要】 莠去津既具有持久性有机污染物的显著特征，又属于环境激素污染物的范畴，是全球比较受关注的污染物之一。除欧盟外，莠去津仍在全球范围内大面积使用，它对土壤生态系统势必产生一定程度的影响。系统地从微生物生物量及土壤微生物的功能、结构和遗传多样性的角度考察其土壤微生物的生态效应，报道较少。本项目以莠去津为污染物，在室内模拟的条件下，开展莠去津对土壤微生物的生态毒理效应研究，采用熏蒸提取的方法确定微生物生物量变化，通过Biolog、T-RFLP和DGGE等技术手段明确莠去津对土壤微生物功能、结构和遗传多样性的影响。将传统方法和分子生物学技术相结合，从生化和分子水平上探明莠去津污染对土壤微生物的生态毒性及土壤微生物对土壤污染的响应过程及时间，为客观评价莠去津对土壤微生物的生态风险提供科学依据和技术支持。主要研究结果如下：1、通过微生物的平板计数实验，研究了土壤中0.1、1、10mg/kg三个莠去津浓度染毒影响下土壤细菌、真菌、放线菌的数量变化，实验表明在莠去津的污染影响下，第7、14、21、28、42天土壤中的细菌、真菌、放线菌都呈现出生长受到抑制的现象。且这种抑制效应随莠去津浓度的增大而增强，表现出良好的剂量-效应关系。采用熏蒸-提取法研究了莠去津对土壤微生物生物量碳的影响，结果表明，在莠去津污染的影响下，土壤微生物生物碳量的变化趋势呈现一定的抑制效应，且与莠去津的处理浓度有关，莠去津浓度越高，抑制效应越明显。土壤微生物生物碳量的响应与莠去津处理浓度间呈现出良好的剂量-效应关系，这和莠去津污染状况下土壤微生物数量的响应变化规律是一致的。2、采用BIOLOG ECO研究了莠去津污染状况下土壤微生物功能多样性的变化，结果表明室内条件下低浓度莠去津处理土壤不会影响土壤微生物的群落的整体活性，在整个实验周期内，相对较高浓度的莠去津（10mg/kg）对土壤微生物的整体活性（AWCD）略有抑制，但在大部分时间内差异不明显。Shannon、Simpson和Mcintosh等三种多样性指数在实验条件下的变化规律基本一致，相对较高浓度（10mg/kg）的莠去津对三个指数均表现出轻微的抑制效应，使土壤微生物群落的丰富度降低，常见物种的优势度和物种的均一性均呈减小的表现。微生物主要利用的碳源种类水平顺序为糖类及其衍生物>代谢中间产物和次生代谢产物>脂类和脂肪酸类>氨基酸及其衍生物。3、采用PCR-DGGE技术研究莠去津污染状况下土壤微生物遗传多样性的变化，本文主要对土壤细菌、真菌和古菌的群落结构的变化规律进行了研究：（1）在莠去津污染对土壤细菌的研究中，通过泳道/条带识别分析、泳道图谱对比分析和聚类分析发现土壤样品中细菌种群受莠去津影响较小，但莠去津的浓度相对越高，其与对照处理组间的相似性和亲缘关系就相对越低；通过计算Shannon、Simpson和Mcintosh多样性指数表明，在整个实验周期内莠去津对土壤细菌的物种丰富度影响甚微，但较高浓度的莠去津却提高了常见菌群的优势度，而低浓度莠去津处理下的细菌的均匀度更好；通过切胶回收条带、连接转化和测序分析后，发现细菌DGGE条带所代表的土壤样品中优势细菌菌群主要属于5个大门类即变形菌门（Proteobacteria）、厚壁菌门（Firmicutes）、栖热菌门（Deinococcus-Thermus）、放线菌门（Actinobacteria）、酸杆菌门（Acidobacteria），其中变形菌类群覆盖了α-Proteobacteria、β-Proteobacteria、γ-Proteobacteria三大菌纲。另外，在莠去津污染条件下，土壤中的厚壁菌门（Firmicutes）的丰度明显增加。（2）在莠去津污染对土壤古菌的研究中，通过泳道/条带识别分析、泳道图谱对比分析和聚类分析发现土壤样品中古菌类群受莠去津影响较小，但莠去津的浓度相对越高，其与对照处理组间的相似性和亲缘关系就相对越低，这与细菌的响应基本一致；通过计算Shannon、Simpson和Mcintosh多样性指数表明，较高浓度的莠去津抑制了古菌类型的丰富度，并提高了常见类型的优势度，而其他处理间差异不显著。通过切胶回收条带、测序分析和构建系统进化树后，发现古菌DGGE条带所代表的土壤样品中优势古菌菌群主要属于3个大门类即泉古菌门（Crenarchaeota）、广古菌门（Euryarchaeota）、Thaumarchaeota。其中有6条条带与氨氧化古菌（AOA）及亚硝化暖菌属（Nitrososphaera）相似度极高,可见莠去津的污染刺激了这些参与氮循环的菌群的活性。（3）在莠去津污染对土壤真菌的研究中，通过泳道/条带识别分析、泳道图谱对比分析和聚类分析，发现相对较高浓度的莠去津对土壤真菌的类群存在轻微的影响；通过计算Shannon、Simpson和Mcintosh多样性指数表明，莠去津的污染对土壤真菌的物种丰富度、最常见物种的优势度已经物种的均一性均未产生明显影响。通过切胶回收条带、测序分析和构建系统进化树后，发现真菌DGGE条带所代表的土壤中真菌的主要类群有子囊菌门（Ascomycota）、担子菌门（Basidiomycota）、不等鞭毛门（Stramenopiles）三大门类。另外，莠去津的存在诱导了真菌中漆斑菌的活性提高或相应丰度的增加。4、采用PCR-T-RFLP技术分析莠去津对土壤中细菌群落结构的影响。结果表明：通过T-RFLP实验，可以比较直观的得到土壤中细菌的优势菌群已经土壤中菌群的变化情况。土壤中细菌在实验莠去津浓度的影响下物种多样性降低，说明在莠去津影响下土壤微生物种群的稳定性受到一定的影响而降低，但除相对较高浓度（10mg/kg）处理的Brillouin指数（H）与Shannon指数（H’）与对照相比变化较显著外（P<0.05），其他各处理组之间无明显差异。将T-RFs数据输入Phylogenetic Assignment Tool数据库进行比对，发现，图谱中占绝对优势地位的T-RFs都可以由属变形菌门（Proteobacteria）、厚壁菌门（Firmicutes）、栖热菌门（Deinococcus-Thermus）、放线菌门（Actinobacteria）、酸杆菌门（Acidobacteria）等门类的菌株16S rDNA酶切产生，这与细菌DGGE测序结果基本吻合。主成分分析（PCA）显示，莠去津的污染并没有对土壤微生物的类群产生较大的影响。更多还原

【Abstract】 With distinct character of permanent organic pollutant, and also belonging toenvironmental hormone pollutants, atrazine has caused great concern for the soil pollution ithas brought. In addition to the European Union, atrazine is still widely used in the worldwhich will have a great influence to soil ecosystem. The present study systematicallyinvestigated the ecological effect of soil microorganism in many aspects from the microbialbiomass, soil microbial function and structure and their genetic diversity, which was lack ofreports so far.Attrazine was selected as objective atrazine for pollutants and the experiment wasperformed to confirm the variation of microbial biomass with the fumigation-extractionmethod, the soil microbes function, structure and the influence of genetic diversity by meansof the Biolog, T-RFLP and DGGE technology, through which to estimate the ecologytoxicological effects of atrazine to soil microorganism in simulating conditions. The presentstudy determined the ecotoxicity of atrazine and the response process and time of soilmicroorganism from biochemic and molecular levels with the combination of traditionalmethods and molecular biology technology and may provide a scientific basis and technicalsupport for ecological risk assessment of attrazine to soil microorganism. The main results areas follows:1. Through the microbial flat count experiment, we studied the quantitative variation ofsoil bacteria, fungi and actinomycetes after exposure to three tested concentrations of attrazine(0.1,1,10mg/kg). The results showed that these three soil microorganism were all inhibitedon days7,14,21,28, and42. And the inhibition exhibited a dose-dependant manner withattrazine enhancement. With the fumigating-extraction method, the effects of soil microbialbiomass carbon were also showed a similar situation after exposed to attrazine.2. The research to varies with functional diversity of soil microorganism under thecondition of attrazine pollution by the systemic BIOLOG ECO method, and the resultssuggested that the indoor conditions deal with low concentration of attrazine would not affectthe whole community of soil microbial activity. In the whole experiment cycle, the whole ofthe soil microbial activity (AWCD) was slightly inhibited with high concentrations of attrazine (10mg/kg), but exhibited no obvious difference for most of time. The similarsituation was also appeared in three diversity indexes including Shannon, Simpson andMcintosh at the same concentration of attrazine. As a result, the soil microbial communitiesrichness, the uniformity of advantage degree of and species were decreased. The main use ofcarbon source microbial species level for sugar were raised and its derivatives order wassubsequently metabolic and secondary metabolites, lipid and fatty acid kind, amino acid andits derivatives.3. The changes of genetic diversity to soil microorganism was investigated by theapplication of by PCR-DGGE technology. The variation of ill the soil bacteria, fungi andbacteria of the community structure are as follows:(1) In the investigation of soil microorganism by attrazine pollution, the lane/striprecognition analysis, lane map contrast analysis and cluster analysis was adopted and wefound that soil microorganism were hardly influenced, while their similarity and relationshipswith control groups were relatively low at high attrazine concentrations; with the calculationof diversity index by Shannon, Simpson Mcintosh, we found that in the whole experimentcycle, soil bacteria the species richness was little influenced, but high concentrations ofattrazine could improve the common advantage of bacterium group and the homogeneousdegree of the bacteria were better at low concentrations; Through the rubber cutting recyclestrip, links, transformation and the sequencing analysis, we found the predominant microbeswhich were represented by bacteria DGGE strip were mainly belong to five categories, theywere Proteobacteria, Firmicutes, Deinococcus-Thermus, Actinobacteria, Acidobacteria.Among Proteobacteria, α-Proteobacteria、β-Proteobacteria、γ-Proteobacteria were covered.In addition, in the attrazine pollution condition, the abundance of Firmicutes was increasedsignificantly.(2) The lane/strip recognition analysis, lane map contrast analysis and cluster analysiswere applied to investigate the Archaea bacteria. The results suggested that the similarity andrelationships were relatively lower compare with control groups with the attrazine increasing,which was in accordance with soil bacteria; with the calculation of diversity index byShannon, Simpson Mcintosh, we found that the bundance of Archaea bacteria was inhibitedwhile the dominance was improved at high attrazine concentrations. Through the rubber cutting recycle strip, links, transformation and the sequencing analysis, we found thepredominant microbes which were represented by bacteria DGGE strip were mainly belong tothree categories, they were Crenarchaeota, Euryarchaeota and Thaumarchaeota, among whichsix led showed a high similarity with ammonium oxidation ancient fungus (AOA) diagramand the nitrosation of warm bacteria (Nitrososphaera) similarity is extremely high. It wasobviously that the atrazin pollution participated in nitrogen cycle of bacterium group activity.(3)The lane/strip recognition analysis, lane map contrast analysis and cluster analysiswas used for the determination of attrazine to soil fungi. We found that relative highconcentrations would have slight effects to the class group. With the calculation of diversityindex by Shannon, Simpson Mcintosh, we found both the abundance of fungal and thedominance of the most common species were exhibited no marked effects. Through therubber cutting recycle strip, links, transformation and the sequencing analysis, we found thepredominant microbes which were represented by bacteria DGGE strip were mainly havethree categories, they were Ascomycota, Basidiomycota and Stramenopiles. In addition, theexistence of attrazine induced the activity or the relevant abundance of Myrothecium.4. The PCR-T-RFLP technology was used to analyse the effects of attrazine to thebacterium structure in soil. The results suggested that the predominant bacterial flora in soilmicroorganisms and their changes could be visualized achieved by means of T-RFLPexperiment. The decreased of the stability of microbial population may be ascribe to thedecrease of species diversity, there were no significant differences in other treatment groupsexcepting for Brillouin index (H) and Shannon index (H’) on10mg/kg concentration attrazine(P<0.05). Contrast to other data in Phylogenetic Assignment Tool, T-RFs which play animportant role in map could be produced by the16S rDNA enzyme of strains such asProteobacteria, Firmicutes, Deinococcus-Thermus, Actinobacteria, and Acidobacteria. Theresults were in accordance with DGGE sequences. By using principal component analysis, nogreat effects on soil microorganisms were found after exposure to attrazine.更多还原