【作者】 李小林；

【导师】 张小平； Kristina Lindstr(o|¨)m；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 为了了解成熟期烟草的根际效应,本文以盐边县代表性烟草种植区域的成熟期烟草根际及非根际土壤为研究对象,测定了主要土壤理化性质,应用化学分析方法研究了微生物生物量碳、土壤酶活性和真菌生物量,利用平板计数法分析了可培养微生物数量,利用16S rDNA限制性片段长度多态性(16S rDNA-RFLP)分子标记法分析了可培养细菌、放线菌、真菌及固氮细菌的群落结构和多样性,结合长度多态片段PCR(LH-PCR)和变性凝胶梯度电泳(DGGE)技术分析了免培养细菌群落结构及多样性,利用DGGE分析了免培养放线菌、真菌(包括18S基因和ITS基因)、nifH基因、硝化细菌及丛枝菌根真菌群落结构和多样性,结合典型对应分析和多元回归树分析研究了土壤环境因子对微生物学特性的影响,进而探讨了成熟期烟草的根际效应。主要结果如下：(1)土壤理化性质分析结果表明,烟草根际在一定程度上有酸化土壤和增加土壤有机质含量的趋势。由于菌根真菌和根际酸化作用的原因,烟草根际总磷、速效磷及速效钾含量显著高于非根际土壤。由于植物根际强烈吸收养分物质等原因,根际土壤中总氮的含量明显低于非根际土壤。(2)对烟草土壤微生物生物量碳、土壤酶活性、真菌生物量、可培养微生物数量、可培养及免培养微生物群落结构和多样性的分析结果表明,由于烟草自毒物质的积累,虽然典型的根际效应在某些样点某些特性中能观察到,但系统的根际效应在成熟期烟草中没有体现。(3)成熟期烟草土壤可培养微生物中细菌遗传多样性最为丰富,其次为可培养真菌和放线菌,固氮细菌最弱。在DGGE分析中,免培养微生物中细菌遗传多样性最丰富,其次依次为放线菌、真菌ITS基因、nifH基因、丛枝菌根真菌、硝化细菌,最弱的为真菌18S基因。(4)在对比实验中,结合LH-PCR和DGGE分析细菌群落结构结果表明,DGGE分析的细菌多样性指数显著高于LH-PCR分析结果,同时所获取的微生物群落结构信息更为丰富,但LH-PCR技术相对于DGGE技术更为简便、快速、稳定和低耗。结合真菌18S基因和ITS基因对比分析结果表明,真菌ITS基因扩增的真菌遗传多样性指数显著高于18S基因。(5)成熟期烟草根际和非根际可培养微生物优势种群差异显著。其中,根际可培养优势细菌为金黄杆菌属(Chryseobacterium)、黄杆菌属(Flavobacterium)、土壤杆菌属(Agrobacterium)、假单胞菌属(Pseudomonas)、寡养单胞菌属(Stenotrophomonas)、肠杆菌属(Enterobacter)和不动杆菌属(Acinetobacter)、优势放线菌均为链霉菌属(Streptomyces);优势真菌为根霉属(Rhizopus)、犁头霉属(Absidia)、赤霉属(Gibberella)、镰刀菌属(Fusarium)、青霉属(Penicillium)、曲霉属(Aspergillus)等；可培养固氮菌主要属于根瘤菌属(Rhizobium)和杆菌属(Arthrobacter)及土壤杆菌属(Agrobacterium)。而非根际可培养优势细菌为节杆菌属(Arthrobacter)、短芽胞杆菌属(Brevibacillus)和不动杆菌属(Acinetobacter);优势放线菌均为链霉菌属(Streptomyces);优势真菌为镰刀菌属(Fusarium)、正青霉属(Eupenicillium)、毛壳菌属(Chaetomium)、拟青霉属(Paecilomyces)及下皮黑孔菌属(Cerrena)等；优势固氮细菌为肠杆菌属(Enterobacter)和杆菌属(Arthrobacter)。(6)总体上看,相对于烟草根际土壤,非根际土壤免培养微生物表现出更强的特异性。其中,烟草成熟期根际特有的微生物为栓菌属(Trametes)和部分不可培养真菌；非根际特有微生物包括假诺卡氏菌(Pseudonocardia)、厄氏菌(Oerskovia)、小单孢菌(Promicromonospora)、蜡蚧菌属(Lecanicillium)、假单孢菌属(Pseudomonas)、青霉属(Penicillium)及部分不可培养真菌和子囊真菌。(7)成熟期烟草根际和非根际免培养微生物优势种群差异显著。其中,根际土壤中优势免培养细菌为Steroidobacter属、硝化螺旋菌属(Nitrospira)、根瘤菌目(Rhizobiales)和a-变形菌；优势免培养放线菌为链霉菌(Streptomyces)和暂未准确分类的免培养放线菌；优势免培养真菌为马拉色菌属(Malassezia)、球囊霉属(Glomus)、镰刀菌属(Fusarium)、角担菌科(Ceratobasidiaceae)、伞菌纲(Agaricomycetes)、散囊菌纲(Eurotiomycetes)及暂未准确确定分类系统的不可培养真菌和子囊真菌；优势nifH基因为固氮捲菌属(Azonexus)、红环菌科(Rhodocyclaceae)细菌及不可培养细菌nifH基因；优势硝化细菌为硝化螺旋菌属(Nitrospira)及暂未准确确定分类系统的不可培养细菌；优势丛枝菌根真菌球囊霉属(Glomus)和暂未准确确定分类的不可培养真菌。而成熟期非根际优势免培养细菌为Steroidobacter属、放线菌目(Actinomycetales)和Chitinophagac eae科；优势免培养放线菌为链霉菌(Streptomyces)、分支杆菌(Mycobacterium)、厄氏菌(Oerskovia)及暂未准确分类的免培养放线菌；优势免培养真菌为蜡蚧菌属(Lecanicillium)、镰刀菌属(Fusarium)、角担菌科(Ceratobasidiaceae)、伞菌纲(Agaricomycetes)和暂未准确确定分类系统的不可培养真菌及子囊真菌；优势nifH基因为固氮捲菌属(Azonexus)、红环菌科(Rhodocyclaceae)细菌及不可培养细菌nifH基因；优势硝化细菌为硝化螺旋菌属(Nitrospira)及暂未准确确定分类系统的不可培养细菌：优势丛枝菌根真菌为球囊霉属(Glomus)。(8)综合典型对应分析和多元回归树分析结果表明,总磷及速效磷对烟草土壤微生物生物量碳、土壤酶活性及真菌生物量的影响最大,速效钾及速效磷是影响可培养微生物数量最关键的环境因子,速效钾、速效磷、速效氮及总氮对免培养微生物多样性的影响显著,其中又以速效钾为最显著因素,同时土壤环境因子中总磷及速效磷对细菌多样性影响最大,速效钾对放线菌和真菌多样性影响最大,速效氮和有机质对nifH基因多样性影响最大,速效磷对硝化细菌和丛枝菌根真菌多样性影响最大。更多还原

【Abstract】 In order to understand the rhizosphere effect of tobacco(Nicotiana tabacum) at the mature stage, the rhizosphere and bulk soil samples were collected from representative sites in the tobacco-growing regions in Yanbian, China. The main soil physical and chemical properties of the samples were analyzed. Chemical analysis method was employed to determine the soil microbial biomass carbon, enzyme activities and fungal biomass. The plate count method was used to determine the viable counts of culturable microorganisms.16S rDNA-RFLP was used to study the diversity and community structure of culturable bacteria, actinobacteria, fungi and azotobacteria. Two culture independent mehods, denaturing gradient gel electrophoresis (DGGE) and length heterogeneity polymerase chain reaction (LH-PCR), were combined to study bacterial community structure and diversity. The community structure and diversity of actinobacteria, fungi (including18S and ITS gene), nifH gene, nitrobacteria and arbuscular mycorrhizal fungi (AMF) were analyzed by DGGE. The canonical correspondence analysis (CCA) and multiple regression tree analysis (MRT) were used to explore the microbiological characteristics associated with environmental variables. The results were combined to explore the rhizosphere effect of tobacco at the mature stage.1. The results of soil physical and chemical properties indicated that rhizosphere soils were acidified and the organic matter contents were higher than that of bulk soils. Total and available phosphorus and available potassium were significantly higher in the rhizosphere than in the bulk soil samples, possibly due to mycorrhizal symbionts and acidification of the rhizosphere soils. However, the decrease of total nitrogen indicated competition for nutrients between the plant roots and microbes.2. The results of soil microbial biomass carbon, enzyme activities, fungal biomass, viable microbial counts and the microbial diversity and community structure indicated that a systematical rhizosphere effect on soil microbial characteristics was not found, although typical rhizosphere effect was evident in some of the samples and characteristics tested, due to the accumulation of autotoxins in the tobacco rhizosphere.3. Among the culturable microorganisms, bacteria showed the richest genetic diversity, followed by actinobacteria, fungi and azotobacteria. The results of DGGE analysis indicated that among the microbes bacteria were genetically most diverse, followed by actinobacteria, fungal ITS gene, nifH gene, AMF, nitrobacteria and fungal18S gene.4. In the comparative experiment, the results of comparative analysis of DGGE and LH-PCR showed that the bacterial diversity index detected by DGGE analysis was significantly higher than the bacterial diversity detected by LH-PCR analysis. At the same time, the DGGE revealed more information on the microbial community structure than LH-PCR. However, the LH-PCR method was more convenient, faster, more stable and cheaper than DGGE. The results of comparative analysis of fungal18S gene and ITS gene showed that the fungal diversity index of ITS gene was significantly higher than that of18S gene.5. The results of sequencing of culturable microbes indicated that the dominant species in rhizosphere soil samples were significantly different to that of bulk soil samples. The dominant bacterial groups in tobacco rhizosphere soil samples were Chryseobacterium, Flavobacterium, Agrobacterium, Pseudomonas, Stenotrophomonas, Enterobacter and Acinetobacter; The dominant actinobacterial group was Streptomyces; The dominant fungal groups were Rhizopus, Absidia, Gibberella, Fusarium, Penicillium and Aspergillus; The dominant azobacterial groups were Rhizobium, Arthrobacter and Agrobacterium. However, the dominant bacterial groups in bulk soil samples were Arthrobacter, Brevibacillus and Acinetobacter; The dominant actinobacterial group was Streptomyces; The dominant fungal groups were Fusarium, Eupenicillium, Chaetomium, Paecilomyces and Cerrena; The dominant azobacterial groups were Enterobacter and Arthrobacter.6. According to the culture independent analyses, the dominant species of microorganisms in bulk soil were more specific than in rhizosphere soil. For example, some groups like Pseudonocardia, Oerskovia, Promicromonospora, Lecanicillium, Pseudomonas, Penicillium, some uncultured fungi and uncultured Ascomycota fungi were only present in bulk soil, while Trametes and some uncultured fungi were only present in the rhizosphere soils.7. The sequencing of microbes detected with culture independent methods indicated that the dominant groups in rhizosphere soil samples were significantly different to those of bulk soil samples. The dominant bacterial groups in rhizosphere soil were Steroidobacter, Nitrospira and some genus of Rhizobiales and a-Proteobacteria; The dominant actinobacterial groups were Streptomyces and uncultured actinobacteria; The dominant fungal groups were Malassezia, Glomus, Fusarium, Ceratobasidiaceae, Agaricomycetes, Eurotiomycetes, uncultured fungi and uncultured Ascomycota fungi; The dominant groups of nifH gene were Azonexus, Rhodocyclaceae and uncultured bacterium; The dominant nitrobacterial groups were Nitrospira and uncultured bacteria; The dominant genus of AMF were Glomus and uncultured fungi. However, the dominant bacterial groups in bulk soil samples were Steroidobacter, Actinomycetales and Chitinophagaceae; The dominant actinobacterial groups were Streptomyces, Mycobacterium, Oerskovia and uncultured actinobacteria; The dominant fungal groups were Lecanicillim, Fusarium, Ceratobasidiaceae, Agaricomycetes, uncultured fungi and uncultured Ascomycota fungi; The domiant species of nifH gene were Azonexus, Rhodocyclaceae and uncultured bacterium; The dominant nitrobacterial groups were Nitrospira and uncultured bacteria; The dominant genus of AMF was Glomus.8. The results of soil microbial diversity associated with environmental variables indicated that total phosphorus and available phosphorus were the major environmental factors affecting the soil microbial biomass carbon, soil enzyme activities and soil fungal biomass. Available potassium and available phosphorus were the main environmental factors affecting the soil viable microbial counts. Available potassium was the most important environmental factor affecting the microbial diversity detected with the culture independent methods, and available phosphorus, available nitrogen and total nitrogen also had definite effects. Total and available phosphorus were the main environmental factors affecting the bacterial diversity detected with the culture independent methods. Available potassium affected actinobacterial and fungal diversity, available nitrogen and organic matter affected nifH gene diversity, and available phosphorus affected the diversity of nitrobacteria and AMF.更多还原