微生物多样性的研究方法概况

介绍了进行微生物多样性研究多种方法,将其简要划分为三大部分：1．经典纯培养技术的改进方法;2．现代分子生物学技术;3．上述两种方法的联合使用;并重点阐述了这些方法的优缺点,展望了微生物多样性研究方法的发展前景。     

Abundance and Diversity of Archaea in Heavy-Metal-Contaminated Soils

The impact of heavy-metal contamination on archaean communities was studied in soils amended with sewage sludge contaminated with heavy metals to varying extents. Fluorescent in situ hybridization showed a decrease in the percentage of Archaea from 1.3% ± 0.3% of 4′,6-diamidino-2-phenylindole-stained cells in untreated soil to below the detection limit in soils amended with heavy metals. A comparison of the archaean communities of the different plots by denaturing gradient gel electrophoresis revealed differences in the structure of the archaean communities in soils with increasing heavy-metal contamination. Analysis of cloned 16S ribosomal DNA showed close similarities to a unique and globally distributed lineage of the kingdom Crenarchaeota that is phylogenetically distinct from currently characterized crenarchaeotal species.     

Integron Diversity in Heavy-Metal-Contaminated Mine Tailings and Inferences about Integron Evolution

Integrons are horizontal gene transfer (HGT) systems containing elements necessary for site-specific recombination and expression of foreign DNA. The overall phylogenetic distribution of integrons and range of genes that can be transferred by integrons are unknown. This report contains an exploration of integrons in an environmental microbial community and an investigation of integron evolution. First, using culture-independent techniques, we explored the diversity of integrons and integron-transferred genes in heavy-metal-contaminated mine tailings. Using degenerate primers, we amplified integron integrase genes from the tailings. We discovered 14 previously undescribed integrase genes, including six novel gene lineages. In addition, we found 11 novel gene cassettes in this sample. One of the gene cassettes that we sequenced is similar to a gene that codes for a step in a pathway for nitroaromatic catabolism, a group of compounds associated with mining activity. This suggests that integrons may be important for gene transfer in response to selective pressures other than the presence of antibiotics. We also investigated the evolution of integrons by statistically comparing the phylogenies of 16S rRNA and integrase genes from the same organisms, using sequences from GenBank and various sequencing projects. We found significant differences between the organismal (16S rRNA) and integrase trees, and we suggest that these differences may be due to HGT.     

Diversity of Arbuscular Mycorrhizal Fungus Populations in Heavy-Metal-Contaminated Soils

High concentrations of heavy metals have been shown to adversely affect the size, diversity, and activity of microbial populations in soil. The aim of this work was to determine how the diversity of arbuscular mycorrhizal (AM) fungi is affected by the addition of sewage-amended sludge containing heavy metals in a long-term experiment. Due to the reduced number of indigenous AM fungal (AMF) propagules in the experimental soils, several host plants with different life cycles were used to multiply indigenous fungi. Six AMF ecotypes were found in the experimental soils, showing consistent differences with regard to their tolerance to the presence of heavy metals. AMF ecotypes ranged from very sensitive to the presence of metals to relatively tolerant to high rates of heavy metals in soil. Total AMF spore numbers decreased with increasing amounts of heavy metals in the soil. However, species richness and diversity as measured by the Shannon-Wiener index increased in soils receiving intermediate rates of sludge contamination but decreased in soils receiving the highest rate of heavy-metal-contaminated sludge. Relative densities of most AMF species were also significantly influenced by soil treatments. Host plant species exerted a selective influence on AMF population size and diversity. We conclude based on the results of this study that size and diversity of AMF populations were modified in metal-polluted soils, even in those with metal concentrations that were below the upper limits accepted by the European Union for agricultural soils.     

西藏扎布耶盐湖细菌多样性的免培养技术分析

【目的】利用免培养技术,获得有关西藏高原高盐度、高海拔盐湖的细菌多样性认识。【方法】从西藏扎布耶盐湖沉积样品中提取微生物总DNA,利用细菌引物f530/r1492扩增16S rRNA基因,然后构建16S rRNA基因质粒文库。采用HaeⅢ和HhaⅠ两种内切酶对阳性克隆质粒DNA进行ARDRA分型分析,根据分型结果挑选克隆进行测序。得到它们的16SrRNA基因部分序列,根据获得的序列构建构建系统发育树。【结果】在系统发育树上,部分克隆(占总克隆数的57.14%)与已知细菌属归于同一分支,主要分布在γ-变形菌纲、α-变形菌纲、δ-变形菌纲、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)和疣微菌门(Verrucomicrobia)的23个嗜盐细菌属之中。其余的克隆为未培养序列,与前者差异很大,在进化树上形成了独立的分支。【结论】研究结果显示出扎布耶茶卡湖中的细菌组成具有极其丰富的多样性。     

Assessment of the Bacterial Diversity in Fenvalerate-Treated Soil

The impact of the pesticide fenvalerate on the diversity of the bacterial community in soil was investigated in this study. After treatment with 0.1, 0.5 or 1.0 mg fenvalerate g^–1 soil in three soils and incubation for a 40-day period, the changes in diversity were monitored by two different methods. The cultivable heterotrophic diversity was investigated by colony morphology on solid LB medium. Genetic diversity was measured as bands on denaturing gradient gel electrophoresis (DGGE) gels by total genomic DNA extraction and purification, PCR-amplification of bacterial 16S rDNA fragments. The Shannon–Wiener index of diversity (H), richness (S) and evenness (E _H) were used to measure changes in the bacterial community in the soils. The results of the cultivable heterotrophic diversity and genetic diversity showed that there was an obvious decrease in diversity due to the application of fenvalerate to the soils, and the different amounts added had different impacts on the diversity. Bands appearing to be either enhanced or inhibited as a result of the fenvalerate treatments were excized and sequenced. Sequencing of excized DGGE bands indicated that application of fenvalerate had an obvious impact on several Pseudomonas spp., or Xanthomonas campestrisor Streptomyces avermitilis. This revealed that microbial community changes can occur due to the application of fenvalerate to soil.     

Exploring the Antibacterial and Antifungal Potential of Jellyfish-Associated Marine Fungi by Cultivation-Dependent Approaches

Fungi isolated from marine invertebrates are of considerable importance as new promising sources of unique secondary metabolites with significant biomedical potential. However, the cultivable fungal community harbored in jellyfish was less investigated. In this work, we seek to recover symbiotic fungi from different tissues of jellyfish Nemopilema nomurai. A total of seven morphotypes were isolated, which were assigned into four genera (Aspergillus, Cladosporium, Purpureocillium, and Tilletiopsis) from two phyla (Ascomycota and Basidiomycota) by comparing the rDNA-ITS sequences with the reference sequences in GenBank. The most fungi were found in the inner tissues of subumbrella. Two of the cultivation-independent procedures, changing media type and co-cultivation, were employed to maximize the complexity of metabolites. Thus, thirteen EtOAc gum were obtained and fingerprinted by High Performance Liquid Chromatography (HPLC) equipped with a photodiode array (PDA) detector. Antibacterial and antifungal activities of these complex mixtures were tested against a panel of bacterial and fungal pathogens. The antimicrobial results showed that all of the 13 EtOAc extracts displayed different levels of antibacterial activity, three of which exhibited strong to significant antibacterial activity to the bacterial pathogens Staphylococcus aureus and Salmonella entrica. Antifungal activity indicated that the EtOAc extracts from pure culture of Aspergillus versicolor and co-culture of A. versicolor and Tilletiopsis sp. in rice media were promising for searching new compounds, with the maximal mycelial growth inhibition of 82.32% ± 0.61% for Rhizoctonia solani and 48.41% ± 11.02% for Botrytis cinerea at 200 μg/ml, respectively. This study is the first report on the antibacterial and antifungal activity of jellyfish-associated fungi and allows the first sight into cultivable fungal community residing in jellyfish. Induced metabolites by cultivation-dependent approaches provides a new reservoir for drug discovery from jellyfish-derived fungi.     

Rapid Method of Determining Factors Limiting Bacterial Growth in Soil

A technique to determine which nutrients limit bacterial growth in soil was developed. The method was based on measuring the thymidine incorporation rate of bacteria after the addition of C, N, and P in different combinations to soil samples. First, the thymidine incorporation method was tested in two different soils: an agricultural soil and a forest humus soil. Carbon (as glucose) was found to be the limiting substance for bacterial growth in both of these soils. The effect of adding different amounts of nutrients was studied, and tests were performed to determine whether the additions affected the soil pH and subsequent bacterial activity. The incubation time required to detect bacterial growth after adding substrate to the soil was also evaluated. Second, the method was used in experiments in which three different size fractions of straw (1 to 2, 0.25 to 1, and <0.25 mm) were mixed into the agricultural soil in order to induce N limitation for bacterial growth. When the straw fraction was small enough (<0.25 mm), N became the limiting nutrient for bacterial growth after about 3 weeks. After the addition of the larger straw fractions (1 to 2 and 0.25 to 1 mm), the soil bacteria were C limited throughout the incubation period (10 weeks), although an increase in the thymidine incorporation rate after the addition of C and N together compared with adding them separately was seen in the sample containing the size fraction from 0.25 to 1 mm. Third, soils from high-pH, limestone-rich areas were examined. P limitation was observed in one of these soils, while tendencies toward P limitation were seen in some of the other soils.     

Phylogenetic Analysis of Microbial Diversity in the Rhizoplane of Oilseed Rape (Brassica napus cv. Westar) Employing Cultivation-Dependent and Cultivation-Independent Approaches

The structure of the microbial rhizoplane community of the important crop plant oilseed rape was studied by using a culture-dependent as well as a culture-independent approach based on 16S rDNA amplification. After isolation of the microbial community from the rhizoplane of oilseed rape (Brassica napus cv. Westar), the collected suspension was divided into two parts. One part was used for cultivation of bacteria onto three different growth media to establish a culture collection. From the other part of the rhizoplane suspension, genomic DNA was isolated and purified. Thereafter, 16S rDNA was amplified by PCR and cloned to obtain a library of 16S rDNA genes representative for the bacterial communities of this habitat. Phylogenetic 16S rDNA sequence analysis of 103 clones of this library revealed considerable differences from the corresponding nucleotide sequences of 111 cultured bacteria. Whereas the 16S rDNA clone library was dominated by a-Proteobacteria and bacteria of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum (51% and 30%, respectively), less than 17% of the cultured bacteria belonged to these two groups. More than 64% of the cultivated isolates were allocated to the b- and g-subclasses of the Proteobacteria, which were present in the clone library at about 14%. Most of the clones of the a-Proteobacteria of the library showed highest similarity to Bradyrhizobium sp. No such bacteria were found in the culture collection. Similarly, the second dominant group of the clone library comprising members of the CFB phylum was represented in the culture collection by a single isolate. The phylogenetic analysis of isolates of the culture collection clearly emphasized the need to use different growth media for recovery of rhizoplane bacteria. Whereas most of the a-Proteobacteria were recovered on complex medium, most of the b-Proteobacteria were isolated onto minimal media. Our results demonstrate that the combined approach pursued in this paper is necessary to explore the biodiversity of bacterial rhizoplane communities.     

乙草胺对农田土壤细菌多样性的影响

在实验室条件下 ,将农田土壤分别用终浓度为 1 0mg、5 0mg和 1 0 0mg g土壤的乙草胺处理 4 0d后 ,检测可培养的异养细菌的多样性和细菌物种多样性。可培养的异养细菌的多样性依据在LB平板上的菌落形态来研究 ,细菌物种多样性则通过基因组DNA的提取 ,纯化 ,1 6SrDNA片段的扩增和变性梯度凝胶电泳 (DGGE)的分离来研究 ,香农多样性指数 (H) ,丰度 (S)和均匀度 (EH)等指标用于评价细菌多样性。实验结果表明 ,与对照土壤相比 ,处理土壤中上述两种类型的细菌多样性均降低 ,而且 ,不同处理浓度对土壤细菌多样性的影响也不同。将DGGE图谱中的条带回收并测序 ,结果显示乙草胺对土壤中的Proteobacteria的α Proteobacteria和γ Proteobacteria影响明显 ,表明乙草胺会在一定程度上改变土壤细菌多样性     

土壤细菌DNA提取及多样性分析的T-RFLP方法

获得高质量的土壤总DNA是土壤细菌生态学的关键步骤之一.实验通过综合应用两个试剂盒(Soilmaster kit和DNA IQTM系统)的优点进行土壤样品总DNA的提取,结果证明该方法是一种快速、有效、灵敏、稳定的土壤DNA提取方法.另外尝试将16S rDNA序列和T-RFLP(Terminal restriction fragment 1ength polymorphism)技术引入土壤细菌DNA群落多样性的研究中,证明T-RFLP是一种有力的土壤细菌多样性分析工具.     

Effects of Plant Biomass, Plant Diversity, and Water Content on Bacterial Communities in Soil Lysimeters: Implications for the Determinants of Bacterial Diversity

Soils may comprise tens of thousands to millions of bacterial species. It is still unclear whether this high level of diversity is governed by functional redundancy or by a multitude of ecological niches. In order to address this question, we analyzed the reproducibility of bacterial community composition after different experimental manipulations. Soil lysimeters were planted with four different types of plant communities, and the water content was adjusted. Group-specific phylogenetic fingerprinting by PCR-denaturing gradient gel electrophoresis revealed clear differences in the composition of Alphaproteobacteria, Betaproteobacteria, Bacteroidetes, Chloroflexi, Planctomycetes, and Verrucomicrobia populations in soils without plants compared to that of populations in planted soils, whereas no influence of plant species composition on bacterial diversity could be discerned. These results indicate that the presence of higher plant species affects the species composition of bacterial groups in a reproducible manner and even outside of the rhizosphere. In contrast, the environmental factors tested did not affect the composition of Acidobacteria, Actinobacteria, Archaea, and Firmicutes populations. One-third (52 out of 160) of the sequence types were found to be specifically and reproducibly associated with the absence or presence of plants. Unexpectedly, this was also true for numerous minor constituents of the soil bacterial assemblage. Subsequently, one of the low-abundance phylotypes (beta10) was selected for studying the interdependence under particular experimental conditions and the underlying causes in more detail. This so-far-uncultured phylotype of the Betaproteobacteria species represented up to 0.18% of all bacterial cells in planted lysimeters compared to 0.017% in unplanted systems. A cultured representative of this phylotype exhibited high physiological flexibility and was capable of utilizing major constituents of root exudates. Our results suggest that the bacterial species composition in soil is determined to a significant extent by abiotic and biotic factors, rather than by mere chance, thereby reflecting a multitude of distinct ecological niches.     

昭苏县野生羊肚菌根际土壤细菌多样性研究

采集昭苏县野生羊肚菌(Morchella esculenta)根际水平直径10 cm、深度0～30 cm范围内的3层各10 cm土壤样品,利用Illumina miseq 2×300 bp高通量测序技术和生物信息学分析方法检测细菌类群的多样性。结果显示,细菌群落Chao和Shannon指数在0～30 cm土层中的平均值分别为152和3.68,且两种指数在10～20 cm土层均最高;细菌群落在各分类水平上的类群(最大类群丰度)分别为变形菌门(Proteobacteria,36.3%)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)等13门;黄杆菌科(Flavobacteriaceae,8.4%)、芽胞杆菌科(Bacillaceae)和葡萄球菌科(Staphylococcaceae)等43科;葡萄球菌属(Staphylococcus,7.1%)、杆状菌属(Bacillus)和Cloacibacterium等52属;不能培养的α-变形杆菌(uncultured α-proteobacterium,2.0%)、不能培养的嗜甲基菌科1种(uncultured Methylophilaceae bacterium)和路西法芽胞杆菌(Bacillus luciferensis)等44种。检测到固氮菌32属,包括15属自生固氮菌、9属共生固氮菌和8属联合固氮菌。结果表明,羊肚菌土壤根际微生物样品中细菌类群具有多样性,10～20 cm土层根际微生物多样性较高,但很多种类丰度较低;该土样含有丰富的固氮菌,这对栽培羊肚菌如何选地或覆土具有一定的指导意义。     

Evaluation of Bacterial Diversity in Soil Microcosms at Different Moisture Contents

The succession analysis of bacterial diversity in the A horizons (rich in organic matter) of three contrasting types of soil—burozem, soddy gley soil, and chernozem—showed that the bacterial diversity of soil microcosms in humid regions can be adequately evaluated only if soil samples are incubated at different soil moisture contents. A complete account of actinobacteria and proteobacteria requires the levels of soil moisture corresponding to the maximum capillary–sorption moisture and capillary moisture, respectively. The bacterial diversity, whose value was maximum on the 40th day of succession, was higher in soddy gley soil than in burozem. The taxonomic structures of the bacterial communities of these two types of soil were different. After wetting chernozem samples from arid regions, the soil bacterial community changed insignificantly with time and drastically differed from that of soils from humid regions. The difference in the bacterial diversity of soils was the most distinct when it was evaluated by measuring the proportion between proteobacteria and actinobacteria.     

Minerals Affect the Specific Diversity of Forest Soil Bacterial Communities

Minerals constitute an ecological niche poorly investigated in the soil, in spite of their important role in biogeochemical cycles and plant nutrition. To evaluate the impact of minerals on the structure of the soil bacterial communities, we compared the bacterial diversity on mineral surfaces and in the surrounding soil. Three pure and calibrated minerals (apatite, plagioclase and a mix of phlogopite-quartz) were buried into the organo-mineral layer of a forest soil. After a 4-year incubation in soil conditions, mineral weathering and microbial colonization were evaluated. Apatite and plagioclase were the only two significantly weathered minerals. The analysis of the 16S rRNA gene sequences generated by the cloning-sequencing procedure revealed that bacterial diversity was higher in the surrounding soil and on the unweathered phlogopite-quartz samples compared with the other minerals. Moreover, a multivariate analysis based on the relative abundance of the main taxonomic groups in each compartments of origin demonstrated that the bacterial communities from the bulk soil differed from that colonizing the minerals. A significant correlation was obtained between the dissolution rate of the minerals and the relative abundance of Beta-proteobacteria detected. Notably, many sequences coming from bacteria colonizing the mineral surfaces, whatever the mineral, harbored high similarity with efficient mineral weathering bacteria belonging to Burkholderia and Collimonas genera, previously isolated on the same experimental site. Taken together, the present results provide new highlights concerning the bacterial communities colonizing minerals surfaces in the soil and suggests that the minerals create true ecological niches: the mineralosphere.     

Diversity Analysis of Bacterial Community from Permafrost Soil of Mo-he in China

The permafrost soil of Mo-he in Northeast China presents a typical cold environment colonized by psychrophilic microorganisms. This study is aimed at assessing the bacterial communities of permafrost soil of Mo-he in China by sequencing the 16S rRNA genes and Mothur analysis. PCR products with universal 16S rRNA gene primers were cloned and partially sequenced, and bacterial identification at the species was performed by comparative analysis with the GenBank/EMBL/DDBJ database. A total of 266 clones were obtained with the average length of 1,050 bp. Mothur analysis showed that the coverage value of clone library was 53.78 %, Shannon diversity (H) was 4.03, Simpson diversity value was 0.018, and 74 operational taxonomic units were generated. Through phylogenetic assignment using BLASTN by more than 97 % similarity, a total of 87 tentative taxa were identified. The majority of bacterial sequences recovered in this study belonged to the Acidobacteria, Proteobacteria, Verrucomicrobia, Bacteroidetes, Chloroflexi and Chlorobi. Among them, Acidobacteria are dominant community, accounting for 30.1 % of total bacteria, followed by Proteobacteria which accounted for 22.2 %. This result reflected the acidic characteristics of the permafrost soil of which pH value was 6.0. Our study indicated that the permafrost soil of Mo-he in China has a high diversity of bacteria and represents a vast potential resource of novel bacteria. As far as we knew, this is the first report on bacterial diversity of permafrost soil of Mo-he in China.     

Determining Diversity of Freshwater Fungi on Decaying Leaves: Comparison of Traditional and Molecular Approaches

Traditional microscope-based estimates of species richness of aquatic hyphomycetes depend upon the ability of the species in the community to sporulate. Molecular techniques which detect DNA from all stages of the life cycle could potentially circumvent the problems associated with traditional methods. Leaf disks from red maple, alder, linden, beech, and oak as well as birch wood sticks were submerged in a stream in southeastern Canada for 7, 14, and 28 days. Fungal biomass, estimated by the amount of ergosterol present, increased with time on all substrates. Alder, linden, and maple leaves were colonized earlier and accumulated the highest fungal biomass. Counts and identifications of released conidia suggested that fungal species richness increased, while community evenness decreased, with time (up to 11 species on day 28). Conidia of Articulospora tetracladia dominated. Modifications of two molecular methods—denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis—suggested that both species richness and community evenness decreased with time. The dominant ribotype matched that of A. tetracladia. Species richness estimates based on DGGE were consistently higher than those based on T-RFLP analysis and exceeded those based on spore identification on days 7 and 14. Since traditional and molecular techniques assess different aspects of the fungal organism, both are essential for a balanced view of fungal succession on leaves decaying in streams.     

辣椒青枯病罹病与健康植株根际土壤微生物群落多样性研究

系统研究和分析辣椒青枯病常发地发病与健康植株土壤微生物群落结构特征,为辣椒青枯病的绿色防治提供理论依据.基于16SrDNA基因高通量测序技术,对辣椒青枯病发病和健康植株根际土壤微生物群落结构和组成进行分析,同时采用biologyeco平板培养技术研究其土壤微生物群落代谢多样性和功能多样性的特征.结果表明,辣椒青枯病发病和健康植株根际土壤微生物群落组成之间存在显著差异,辣椒青枯病发病土壤的OTU为4566个,辣椒青枯病健康土壤的OTU为4167个.依据OTU所属细菌物种信息对土壤细菌群落结构进行分析,变形菌门在发病和健康土壤中均为优势细菌类群,其次为放线菌门类群.其中健康植株根际土壤中芽单胞菌门(Gemmatimonadetes)、装甲菌门(Armatimonadetes)的相对丰度比发病植株的分别高出了4.37,3.87倍,而发病植株根际土壤中厚壁菌门(Firmicutes)的相对丰度比健康植株的高出了3.87倍.辣椒青枯病发病土壤和健康土壤的土壤微生物代谢多样性也存在显著差异,同时,健康土壤中其微生物群落代谢得到显著增强,特别是对酚类化合物的利用显著增多,对辣椒抗病性存在显著的影响.研究表明,辣椒青枯病发病和健康植株根际土壤微生物群落组成和结构之间存在显著差异,并且健康土壤的微生物群落对酚类化合物的利用显著增强.