【作者】 周慧；

【导师】 高必达； 李迪强；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2008， 博士

【摘要】 土壤微生物是生态系统的重要成员,它参与生态系统的物质循环和能量流动,是土壤中最活跃的部分,它们在土壤中的数量与分布一定程度上反映出土壤肥力状况与植物营养的密切关系,同时也反映土壤、植被和气候等综合因素对土壤微生物的影响。因此土壤微生物多样性的保护和研究对系统生态保护具有重要意义。高黎贡山国家级自然保护区位于云南西部中缅边境地区,海拔760-3100m,具有高山、亚高山生物气候垂直带谱自然景观和异常丰富的生物多样性,是中国生物多样性保护具有全球意义的关键区域,被喻为“世界物种基因库”。研究高黎贡山土壤微生物群落结构与功能,揭示其生态分布和区域特异性,为评价高黎贡山地区的生态环境、了解全球变化对高黎贡山地区土壤微生物的影响以及高黎贡山自然保护区保护策略的制定提供科学依据。本研究采用传统分离培养方法对高黎贡山生物多样性固定监测样地的土壤中主要微生物类群的数量进行研究,探讨其与土壤酶和生态因子的相关性;采用木端限制性酶切片段长度多态性分析(T—RFLP)和基因芯片(GeoChipⅡ)等分子生物学技术对土壤细菌群落、氮循环相关基因(nifH和nosZ)、降解纤维素真菌及11类功能基因的多样性及分布特点进行研究。分离培养了土壤中高效降解纤维素真菌并对其进行分子鉴定。同时探讨了不同植被和海拔高度土壤微生物群落结构与功能的多样性及其主要影响因子。其主要研究结果如下:(1)对高黎贡山海拔960—2 878m的8个生物多样性固定监测样地的土壤微生物进行培养分析,所有样地中细菌数＞放线菌数＞真菌数,细菌、真菌、放线菌数量随海拔不同而变化,各样地间差异极显著(P＜0.05),最高点均出现在海拔2 000m左右的森林样地;好气性纤维素菌和嫌气性纤维素分解菌在低海拔的灌丛和咖啡林数量丰富,而高海拔森林样地中数量相对较低,好气性自生固氮菌和嫌气性自生固氮菌数量在玉米地最高,咖啡林最低,纤维素菌和固氮菌数量在样地间均差异显著(P＜0.05)。土壤微生物量碳在玉米地、咖啡林和海拔2 000m的森林样地中偏高且所有样地间差异极显著(P＜0.01)。土壤微生物随海拔高度变化且与土壤酶及生态因子呈现不同程度的相关性。细菌数、总菌数、微生物量碳分别与脲酶和淀粉酶活显著相关(P＜0.05);真菌数与淀粉酶活显著相关(P＜0.05);嫌气固氮菌与脲酶、蛋白酶、淀粉酶活都显著相关(P＜0.05)。细菌数、微生物总数与土壤有机质含量显著相关(P＜0.05),放线菌数与全氮、全磷、全钾、海拔、含水量、pH值及温度显著相关(P＜0.05);纤维素菌与温度、海拔、含水量显著相关(P＜0.05);好气固氮菌与全磷显著相关(P＜0.05),嫌气固氮菌与有机质、全氮、全磷及含水量显著相关(P＜0.05);微生物量碳与有机质含量显著相关(P＜0.05)。说明影响高黎贡山土壤微生物数量和多样性垂直分布的主要生态因子是土壤酶、温度、有机质、含水量。人为耕作活动和土地利用方式的改变、植被类型等因素对微生物量有重要影响,且这种影响具有综合效应。(2)本研究首次采用核糖体DNA基因文库技术与T-RFLP技术相结合,分析高黎贡山8个不同海拔和植被类型的生物多样性固定监测样地中土壤细菌16S rDNA基因多样性。8个样地Msp I酶切共得到289个OTU,Rsa I酶得到216个OTU。基于Msp I酶切指纹图谱:海拔2 000m左右的森林土壤细菌多样性普遍高于其它海拔和植被的样地,样地内细菌多样性与可培养微生物数量显著相关。16S rDNA文库随机测序150个阳性克隆得到132条特异序列,它们分属细菌的7个门,其中酸杆菌门占总鉴定数的57%,为高黎贡山地区土壤细菌的优势菌群。土地利用方式的不同和温度是影响此地区土壤细菌多样性的主要因素。地理距离和植被类型的差异是影响此地区土壤细菌生态分布的主要因素。此外,土壤pH值可能对群落结构起重要作用,而其它生态因子均与土壤细菌群落结构多样性及生态分布有一定相关。(3)为了解高黎贡山地区土壤氮循环微生物的群落结构及其差异,利用T-RFLP技术对高黎贡山地区土壤微生物固氮酶基因nifH和反硝酶基因nosZ多样性及生态分布进行研究,研究选取了来自于不同海拔高度、植被类型的8个生物多样性固定监测样地。利用4种限制性内切酶(HaeⅢ、HhaⅠ、#saⅠ和MspⅠ)分别对这两个基因进行T—RFLP分析,两个基因均是用HhaⅠ酶切得到OTU数最多:8个样地共得到63个nifH基因OTU,156个nosZ基因OTU。样地内nifH基因多样性为0.454(样地10)—1.200(样地5),各样地均有3—4个优势菌群,nifH基因多样性与土壤pH值相关性较高,与其它生态因子有不同程度相关,受到土壤耕作制度、植被类型等诸多因素的影响;样地内nosZ基因多样性为0.760(样地10)—1.464(样地13),各样地均有2—4个优势菌群,nosZ基因多样性与温度、海拔、pH值、土壤含水量有较强相关。地理距离和植被类型对nifH和nosZ基因的分布有重要影响。(4)采用刚果红平板和PDA平板对研究样地土壤中降解纤维素类真菌进行分离培养,通过液体发酵测定其纤维素酶活差异,筛选相对酶活较高的菌株进行形态学和分子生物学鉴定,共得到35株具有较高纤维素酶活真菌,其96h液体发酵测量CMC酶活力范围在79.6(菌株6)-228.9IU(菌株3)。这些株菌分属15个属,其中4株归属于担子菌类(Basidiomycetes)的3个属,占筛选菌株数的11.4%,31株归属于子囊菌类(Ascomycetes)12个属,占筛选菌株数的88.6%。其中曲霉属(Aspergillus)、青霉属(Penicillium)、葡萄状穗霉属(Sttachybotrys)、毛壳霉属(Chaetomium)的真菌共计20株,占总分离菌株数的57.1%,是高黎贡山土壤中降解纤维素类真菌的优势菌群。其中菌株3被鉴定为扩展青霉,其在酶系和对底物利用都有突出特点,具有开发前景。(5)利用功能基因芯片(GeoChipⅡ)检测高黎贡山高黎贡山海拔960—2 878m的6个样地土壤微生物与碳、氮、磷、硫、磷循环、金属抗性,生物降解相关的11类功能基因。6个样地共检出功能基因1 515个及未知基因27个,各类功能基因检出数:ORG＞MET＞CDEG＞NRED＞NIT＞DSR＞methane ox＞CFIX＞NFIX＞methane gen＞PER。样地检出功能基因数目及多样性:样地5＞样地10＞样地7＞样地13＞样地15＞样地8;土壤微生物功能基因多样性受人为干扰,植被类型、海拔、土壤生态因子等多方面因素共同影响,其中人为干扰和植被类型的变化明显改变了土壤微生物功能群。重点分析了高黎贡山6个样地土壤微生物与碳、氮循环相关的7类基因:CDEG、CFIX、methane ox、methane gen、NFIX、NIT、NRED。这些基因在各样地检出数、多样性和均匀度均不相同,并且同一类基因在各样地的检出情况也互不相同。碳氮循环基因多样性与土壤有机质、全氮、碳氮比、速效磷和土壤含水量等生态因子有不同程度的相关。不同强度的人为干扰和不同的植被类型,对土壤微生物碳氮循环基因种类与分布有显著差异。更多还原

【Abstract】 Soil microorganism play integral and often unique roles in global material cycle of carbon, nitrogen,and so on,thus it affects the material balance,the composition of the atmosphere and geochemistry procedure in the global scale.Soil microbial diversity may represent the ability of a soil to cope with perturbations,and has been proposed as an indicator for soil quality. Understanding the structure and composition of microbial communities and their responses to environmental perturbations,such as toxic contamination,climate change,and land use changes, is critical for prediction,maintenance,and restoration of desirable ecosystem functions.Due to the extremely high diversity of environmental samples,microbial detection,characterization,and quantification are great challenges.The development and application of nucleic acid-based techniques have largely eliminated the reliance on cultivation-dependent methods for microbial detection and consequently have greatly advanced characterization of microorganisms in natural habitats.Gaoligongshan national natural reserve located in the southwestern part of Yunnan Province with altitude ranging from 760 to 3 100m.It is one of the key areas for biodiversity protection in the globe and was named "The Genebank of Earth" for its various species.The forest destruction and the global change caused the environmental deterioration in Gaoligongshan national natural reserve.Research on the distribution and regional character of soil microorganisms in this area could help to evaluate environment,understand the influence in global change and offer scientific data for making the protected policy.In this study,cultured and uncultured methods combined with advanced molecular technology such as microarray,T-RFLP and sequencing were used to investigate the diversity and community structure of microbe,bacteria,cellulose-decomposing fungi and functional genes. The influences of plant community type,altitude and environment factors were discussed.The key results from these studies are summarized as follows:(1) The quantities of soil microbe were studied in different vegetation types and altitudes in Gaoligongshan national natural reserve.The quantities of microbes,the activities of enzymes and the amount of nutrition were examined in 8 soils,which were collected from 8 fixed monitoring sample plots with different habitats and the elevation ranging from 960 to 2 878 meters.The results showed that the quantities of bacteria,actinomyces and fungi varied with elevation.The highest values occurred at the elevation of about 2 000 meters.The quantities of aerobic and anaerobic cellulose-decomposing fungi were plentiful in the scrub and coffee forest with low altitude,while the quantities were relatively few in the plot of primitive forest with high altitude.The quantities of aerobic and anaerobic nitrogen-fixing bacteria were the most in maize plot and the lowest in coffee plot.Therefore,the quantities of cellulose fungi and nitrogen-fixing bacteria were very different in different plots(P＜0.05).Microbial biomass carbon in plots of maize,coffee and forest with the altitude of 2000m were quite plentiful,and the differences among all plots were significant(P＜0.01).The change of soil microorganism with the altitude was relative to the enzyme and ecological factors in the soil.The bacteria quantity,the quantity of total microbe,and the microbial biomass carbon were significantly correlative to urease and amylase respectively(P＜0.05);the quantity of fungi was correlative to amylase significantly(P＜0.05);the anaerobic nitrogen-fixing bacteria was correlative to urease,protease and amylase significantly(P＜0.05).The bacteria quantity and the quantity of total microbe were correlative to the contents of soil organic matters(P＜0.05), and the quantity of actinomycetes was correlative to total nitrogen,total phosphorus,total potassium,altitude,water content,pH vale and temperature(P＜0.05);cellulose-decomposing fungi was correlative to temperature,altitude and water content(P＜0.05);aerobic nitrogen-fixing bacteria was in positive correlation to total phosphorus(P＜0.05),and anaerobic nitrogen-fixing bacteria was also in positive correlation to organic matters,total nitrogen,total phosphorus and water content(P＜0.05);carbon content was positively correlative to content of organic matters (P＜0.05).All results suggested that the major ecological factors influencing on the quantity of soil microorganism and the vertical distribution of diversity were soil enzyme,temperature,soil organic matters and water content.The human cultivating activities,the change of soil utilization and the vegetation type also influenced the quantity of microorganism with the comprehensive effects.(2) Genetic diversity of 16s rDNA of soil bacteria in 8 plots with different altitudes and vegetations in Gaoligongshan national natural reserve was analyzed by the combination technology of ribosome DNA gene library and T-RFLP.There were 289 OTU restricted by MspⅠ,and 216 OTU by Rsa I in 8 plots.Resulting from the fingerprint map of MspⅠ,the diversity of soil bacteria with the altitude about 2000m was more than other plots with different altitudes and vegetations generally,and the bacteria diversity of plot was significantly correlative to the quantity of cultured microorganism.By random sequencing of 16s rDNA library,there were 132 special sequences from 150 positive clones,which belonged to seven phylums.Phylum Acidobacteria accounted for the 57%of total sequenced,which was the dominant bacteria in Gaoligongshan national natural reserve.The different land use and temperature were the major factors influencing on the diversity of soil bacteria.The geological distance and difference on vegetation type were the factors influencing on the ecological distribution of soil bacteria. Moreover,pH value of soil might play important role on the community structure,while other ecological factors were correlative to the diversity of community structure of soil bacteria and ecological distribution.(3) In order to investigate the community structure and difference of microbes related to nitrogen cycle in soil from Gaoligongshan national natural reserve,T-RFLP technology was applied for the analysis on the diversity and ecological distribution of nifH and nosZ and 8 samples with different altitudes and vegetations were selected for this study.There were four restriction enzymes(HaeⅢ,HhaⅠ,RsaⅠ,MspⅠ) applied on nifH and nosZ genes.The number of OTU was the most by restriction of HhaⅠ,63 OTU in nifH and 156 OTU in nosZ respectively.The diversity of nifH(H’) was ranged from 0.454(Plot 10) to 1.200(Plot 5).There were 3-4 dominant floras in each plot.The diversity of nifH was highly correlative to pH value of soil,relative to other ecological factors with different degrees,and was influenced by soil cultivation system and vegetation type.The diversity of nosZ(H’) was ranged from 0.760(Plot 10) to 1.464(Plot 13).There were 2-4 dominant floras in each plot.The diversity of nosZ was highly correlative to temperature,altitude,pH value and water content.The geological distance and vegetation type also greatly influenced the distribution of nifH and nosZ.(4) In order to understand the diversity of culturable cellulose-decomposing fungi(CCDF), Carboxymthyl cellulose-congo red(CMC-CR) plate and potato dextrose agar(PDA) plate were used to isolate and culture the cellulose-decomposing fungi from plots.The quantities and diversity of soil culturable cellulose-decomposing fungi are significantly correlated with organic matter and total nitrogen in the soil.Different plant community and human disturbance have effect on the quantities and diversity of CCDF.There were 35 strains with high activities,the CMC activity by 96h of liquid fermentation was ranged from 79.6 IU(strain 6) to 228.9 IU (strain 3).A total of thirty five independent isolates belongs to 15 genus,which are identified using both morphological characteristics and rDNA ITS sequence analysis.Thirty one(88.6%) isolates belongs to 12 genus from phylum Ascomycetes and four(11.4%) isolates belongs to 3 genus from the phylum Basidiomycetes.Aspergillus,Penicillium,Stachybotrys and Chaetomium were the dominant genus.Strain 3 was identified as the Penicillium expansuma,with great developmental potential since of its particular enzyme system and utilization on substrate.(5) GeoChipⅡwas applied in the detection of functional microorganism of 6 plots on the altitude of 960-2 878m of Gaoligongshan national natural reserve,which including 11 kinds of functional genes,such as carbon cycle,nitrogen cycle,phosphor cycle,sulfur cycle,metal resistance,organic degradation.There were 1 515 functional genes and 27 unknown genes detected out in 6 plots.The order of number of detected genes was ORG＞MET＞CDEG＞NRED＞NIT＞DSR＞methane_ox＞CFIX＞NFIX＞methane_gen＞PER.The order of plots according to the number of genes and diversity was Plot 5＞Plot 10＞Plot 7＞Plot 13＞Plot 15＞Plot 8.The diversity of microorganism functional genes was influenced by various factors including human disturbance,vegetation type,altitude and ecological factors,among them,the human disturbance and the change of vegetation type greatly altered the functions of microorganism.Seven kinds of genes related to carbon and nitrogen cycles in microorganism of 6 plots in Gaoligongshan national natural reserve were analyzed,including CDEG,CFIX,methane_ox, methane_gen,NFIX,NIT and NRED.The number of genes detected out,diversity and evenness of these genes were different in different plots,and the number of the same gene detected from different plot are different.Genetic diversity of carbon-nitrogen cycle was relative to soil organic matters,total nitrogen,carbon-nitrogen ratio,available phosphor and water content with different degrees.The human disturbance with different strength and the different vegetation type also influenced the number and distribution of carbon and nitrogen cycle genes.更多还原