Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of gamma-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

种子微生物生态学研究进展

植物种子微生物生态学是研究与种子相联合的微生物的组成﹑功能﹑演替、它们之间关系及其与宿主之间相互关系的科学。种子中蕴含着丰富的微生物资源,它们对种子以及植物的健康具有重要的影响。不同种类植物种子联合的微生物群落由于受到种子本身及外界环境因素的影响而有所差异。论述了种子微生物生态学的概念、主要研究方法、种子微生物生态系统中的微生物种类、相关影响因素,以及种子微生物生态学研究的发展方向。种子微生物生态学的研究对生产实践有重要意义,同时也将丰富种子生物学的内容,对种子科学的发展起到促进作用。     

Application of quantitative real-time PCR for enumeration of total bacterial,archaeal, and yeast populations in kimchi

Kimchi is a Korean traditional fermented food made of brined vegetables, with a variety of spices. Various microorganisms are associated with the kimchi fermentation process. This study was undertaken in order to apply quantitative real-time PCR targeting the 16S and 26S rRNA genes for the investigation of dynamics of bacterial, archaeal, and yeast communities during fermentation of various types of kimchi. Although the total bacterial and archaeal rRNA gene copy numbers increased during kimchi fermentation, the number of yeasts was not significantly altered. In 1 ng of bulk DNA, the mean number of rRNA gene copies for all strains of bacteria was 5.45×10⁶ which was 360 and 50 times greater than those for archaea and yeast, respectively. The total gene copy number for each group of microorganisms differed among the different types of kimchi, although the relative ratios among them were similar. The common dominance of bacteria in the whole microbial communities of various types of kimchi suggests that bacteria play a principal role in the kimchi fermentation process.     

Microflora profiling of infected root canal before and after treatment using culture-independent methods

This study aimed to profile the microflora in infected root canals before and after root canal treatment using culture-independent methods. Six infected root canals in single-rooted teeth with periapical lesions from five subjects were included. Quantification of total bacteria was performed by real-time PCR with primers targeting 16S rRNA genes. PCR products with universal 16S rRNA gene primers were cloned and partially sequenced, and bacterial identification at the species level was performed by comparative analysis with the GenBank database. The concentration of extracted DNA before treatment was higher than that after root canal treatment, although the difference was not statistically significant. Sequence analysis revealed that oral bacteria such as Fusobacterium, Streptococcus, Olsenella, and Pseudoramibacter detected in cases before root canal treatment disappeared after treatment. These results suggest that the root canal microflora are distinct before and after root canal treatment, and that treatment changes the microflora in both quantity and quality.     

垃圾填埋场中厌氧真菌18SrDNA的PCR扩增及鉴定

采用机械破壁法直接从来自7个不同地区的垃圾填埋场滤液样本中提取真菌DNA,应用真菌通用引物NS1和NS8扩增18SrDNA（约1800bp),多聚酶链式反应（PCR）产物的琼脂糖凝胶电泳结果表明所有的样本均得到了扩增;以PCR产物作为模板,采用厌氧真菌Chytridiomycetes科的专用引物Chyt-719和Chyt-1553进行二次PCR扩增（约857bp),该阳性扩增产物克隆和测序结果首次表明在食草动物瘤胃中存在的厌氧真菌Chytridiomycetes也存在于垃圾填埋场中,且为Neocallimastix属。     

Succession of Microbial Communities during Hot Composting as Detected by PCR–Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4–V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8–V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of γ-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

Bacterial origin and community composition in the barley phytosphere as a function of habitat and presowing conditions

An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.     

Influence of corn,switchgrass, and prairie cropping systems on soil microbial communities in the upper Midwest of the United States

Because soil microbes drive many of the processes underpinning ecosystem services provided by soils, understanding how cropping systems affect soil microbial communities is important for productive and sustainable management. We characterized and compared soil microbial communities under restored prairie and three potential cellulosic biomass crops (corn, switchgrass, and mixed prairie grasses) in two spatial experimental designs – side‐by‐side plots where plant communities were in their second year since establishment (i.e., intensive sites) and regionally distributed fields where plant communities had been in place for at least 10 years (i.e., extensive sites). We assessed microbial community structure and composition using lipid analysis, pyrosequencing of rRNA genes (targeting fungi, bacteria, archaea, and lower eukaryotes), and targeted metagenomics of nifH genes. For the more recently established intensive sites, soil type was more important than plant community in determining microbial community structure, while plant community was the more important driver of soil microbial communities for the older extensive sites where microbial communities under corn were clearly differentiated from those under switchgrass and restored prairie. Bacterial and fungal biomasses, especially biomass of arbuscular mycorrhizal fungi, were higher under perennial grasses and restored prairie, suggesting a more active carbon pool and greater microbial processing potential, which should be beneficial for plant acquisition and ecosystem retention of carbon, water, and nutrients.     

变性梯度凝胶电泳(DGGE)在微生物生态学中的应用

由于从环境样品中分离和培养细菌的困难,分子生物学方法已发展用来描述和鉴定微生物群落。近年来基于DNA方法的群落分析得到了迅速的发展,如PCR扩增技术,克隆文库法,荧光原位杂交法,限制性酶切片段长度多态性法,变性和温度梯度凝胶电泳法。DGGE已广泛用于分析自然环境中细菌、蓝细菌,古菌、微微型真核生物、真核生物和病毒群落的生物多样性。这一技术能够提供群落中优势种类信息和同时分析多个样品。具有可重复和容易操作等特点,适合于调查种群的时空变化,并且可通过对切下的带进行序列分析或与特异性探针杂交分析鉴定群落成员。DGGE分析微生物群落的一般步骤如下：一是核酸的提取,二是16S rRNA,18S rRNA或功能基因如可容性甲烷加单氧酶羟化酶基因(mmoX)和氨加单氧酶a一亚单位基因(amoA)片段的扩增,三是通过DGGE分析PCR产物。DGGE使用具有化学变性剂梯度的聚丙烯酰胺凝胶,该凝胶能够有区别的解链PCR扩增产物。由PCR产生的不同的DNA片段长度相同但核苷酸序列不同。因此不同的双链DNA片段由于沿着化学梯度的不同解链行为将在凝胶的不同位置上停止迁移。DNA解链行为的不同导致一个凝胶带图案,该图案是微生物群落中主要种类的一个轮廓。DGGE使用所有生物中保守的基因片段如细菌中的16S rRNA基因片段和真菌中的18S rRNA基因片段。然而同其他分子生物学方法一样,DGGE也有缺陷,其中之一是只能分离较小的片段,使用于系统发育分析比较和探针设计的序列信息量受到了限制。在某些情况下,由于所用基因的多拷贝导致一个种类多于一条带,因此不易鉴定群落结构到种的水平。此外,该技术具有内在的如单一细菌种类16S rDNA拷贝之间的异质性问题,可导致自然群落中微生物数量的过多估计。DGGE是分析微生物群落的一种有力的工具。不过为了减少DGGE和其它技术的缺陷,建议研究者结合DGGE和其它分子及微生物学方法以便更详细的观察微生物的群落结构和功能。     

Bacterial Origin and Community Composition in the Barley Phytosphere as a Function of Habitat and Presowing Conditions

An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.     

Influence of seed and root exudations on the rhizosphere effect inSorghum vulgare andCrotalaria juncea

The influence of seed and root exudations on the rhizosphere effect ofSorghum vulgare andCrotalaria juncea was examined. While the quantitative occurrence of certain morphological and physiological groups of bacteria on the seeds and rhizospheres of the two plant species differed, there were similarities in the occurrence of certain physiological and nutritional groups of bacteria on the seed as well as on the rhizosphere. The occurrence of some genera of fungi on the seeds and in the rhizosphere indicated the influence of both seeds and root exudations on the rhizosphere effect. Increase in root exudations of amino acids and sugars correlated with a concomitant increase in microbial activity in the rhizosphere on the 15th day of growth in both plant species but there was a delayed effect of the exuded chemicals on the rhizosphere microflora after 30 d of plant growth. Based on the Ph.D. thesis of the first author submitted to the University of Agricultural Sciences, Bangalore.     

3 次连续重复提取DNA 能较好反映土壤微生物丰度

【目的】研究同一个土壤需要反复提取几次才能在最大程度上反映土壤微生物的丰度,探讨风干土壤代替新鲜土壤用于微生物丰度研究的可行性。【方法】针对两种理化性质具有较大差异的旱地和稻田新鲜土壤及其风干土壤,分别对土壤微生物进行5次连续裂解提取DNA。通过实时荧光定量PCR技术分析连续反复提取对土壤古菌和细菌16S rRNA gene数量、氨氧化古菌和细菌功能基因amoA数量的影响。【结果】3次连续提取DNA占5次提取DNA总量的76%以上,氨氧化古菌、氨氧化细菌、古菌和细菌4类微生物的3次连续提取最低回收率为77.5%;与新鲜土壤相比,风干处理导致氨氧化古菌、氨氧化细菌、古菌、细菌的数量分别降低84.3%、81.2%、12.5%和90.3%,然而,2种土壤风干过程中主要微生物类群的数量变化规律基本一致,表明土壤微生物对风干处理的响应可能受土壤类型的影响较小。【结论】土壤微生物连续3次裂解能较好反映微生物丰度。与新鲜土壤相比,风干过程显著降低了土壤微生物丰度,然而,通过风干土壤中微生物丰度的变化趋势反映新鲜土壤中微生物数量变化规律具有一定的可行性。     

生物造粒流化床微生物落结构及其动态变化

为了研究生物造粒流化床污水处理反应器颗粒污泥中微生物群落结构及其动态变化,分别从生物造粒流化床10、60、110cm处取颗粒污泥,通过细胞裂解直接提取颗粒污泥细菌基因组DNA。以细菌和古细菌16S rRNA基因通用引物530F/1490R,对活性污泥中提取的细菌基因组DNA进行PCR扩增,长约1kb的PCR扩增产物纯化后经变性梯度凝胶电泳(DGGE)分离,获得微生物群落的DNA特征指纹图谱。结果显示,生物造粒流化床反应器颗粒污泥中的微生物群落非常丰富,在10cm处微生物的种属达到23种,60cm处为21种,110cm处为20种;生物造粒流化床不同高度都有一些各自的特有种属和共有种属,反应器不同高度的微生物群落演替不明显,微生物群落相似性为83.1%,群落结构较为稳定。     

Analysis of Bacterial and Fungal Communities in Japanese- and Chinese-Fermented Soybean Pastes Using Nested PCR–DGGE

The microbial diversity of Japanese- and Chinese-fermented soybean pastes was investigated using nested PCR–denaturing gradient gel electrophoresis (DGGE). Five Japanese-fermented soybean paste samples and three Chinese-fermented soybean paste samples were analyzed for bacteria and fungi. Extracted DNA was used as a template for PCR to amplify 16S rRNA and 18S rRNA genes. The nearly complete 16S rRNA and 18S rRNA genes were amplified using universal primers, and the resulting products were subsequently used as a template in a nested PCR to obtain suitable fragments for DGGE. Tetragenococcus halophilus and Staphylococcus gallinarum were found to dominate the bacterial microbiota in Japanese samples, whereas Bacillus sp. was detected as the predominant species in Chinese samples. DGGE analysis of fungi in soybean pastes determined the presence of Aspergillus oryzae and Zygosaccharomyces rouxii in most of the Chinese and Japanese samples. Some differences were observed in the bacterial diversity of Japanese- and Chinese-fermented soybean pastes.     

Tomato genotype and Azospirillum inoculation modulate the changes in bacterial communities associated with roots and leaves

AIMS: To evaluate the effect of plant variety and Azospirillum brasilense inoculation on the microbial communities colonizing roots and leaves of tomato (Lycopersicon esculentum Mill.) plants. METHODS AND RESULTS: Seeds of cherry and fresh-market tomato were inoculated with A. brasilense BNM65. Sixty days after planting, plants were harvested and the microbial communities of the rhizoplane and phyllosphere were analysed by community-level physiological profiles (CLPP) using BIOLOG EcoPlates and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Differences on the rhizoplane and phyllosphere bacterial communities between the two tomato types were detected by principal component analysis of the CLPP; DGGE fingerprints also showed differences at the phyllosphere level. Fresh-market tomato had a more complex phyllosphere bacterial community than cherry tomato, as determined by DGGE profiles. Physiological and genetic changes on phyllosphere and rhizoplane bacterial communities by Azospirillum seed inoculation were evident only on cherry tomato. CONCLUSIONS: Tomato genotype affects the response of native bacterial communities associated with the roots and leaves to A. brasilense seed inoculation. SIGNIFICANCE AND IMPACT OF THE STUDY: The successful implementation of Azospirillum inoculation requires not only the consideration of the interactions between A. brasilense strains and plant genotypes, but also the plant-associated microflora.     

Influence of nitrate—ammonium ratio on growth and nutrition of Arabidopsis thaliana

We investigated the microbial community structure and population size of arboreal soils—including canopy and bromeliad epiphytic leaf-tank soils—and ground soils in a tropical lowland rainforest in Costa Rica using molecular and cultivation methods. PCR-DGGE analysis of 16S rRNA and 18S rRNA gene fragments and quantitative real-time PCR were applied to survey the bacteria, ammonia-oxidizing bacteria (AOB), and fungi. Bacteria from epiphytic tank soils were isolated and identified. Bacillaceae, Pseudomonadaceae and Micrococcaceae were the most abundant families. According to cluster analysis of DGGE fingerprints a significant difference among the three soil types was evident for bacterial communities. In addition, the microbial communities of canopy and tank soils clustered apart from ground soils. The fungal and AOB communities were diverse but non-specific for the soil types analyzed.     

Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization

Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities.     

Seasonal changes in nitrogen-cycle gene abundances and in bacterial communities in acidic forest soils

The abundance of genes related to the nitrogen biogeochemical cycle and the microbial community in forest soils (bacteria, archaea, fungi) were quantitatively analyzed via real-time PCR using 11 sets of specific primers amplifying nifH, bacterial amoA, archaeal amoA, narG, nirS, nirK, norB, nosZ, bacterial 16S rRNA gene, archaeal 16S rRNA gene, and the ITS sequence of fungi. Soils were sampled from Bukhan Mountain from September of 2010 to July of 2011 (7 times). Bacteria were the predominant microbial community in all samples. However, the abundance of archaeal amoA was greater than bacterial amoA throughout the year. The abundances of nifH, nirS, nirK, and norB genes changed in a similar pattern, while narG and nosZ appeared in sensitive to the environmental changes. Clone libraries of bacterial 16S rRNA genes were constructed from summer and winter soil samples and these revealed that Acidobacteria was the most predominant phylum in acidic forest soil environments in both samples. Although a specific correlation of environmental factor and gene abundance was not verified by principle component analysis, our data suggested that the combination of biological, physical, and chemical characteristics of forest soils created distinct conditions favoring the nitrogen biogeochemical cycle and that bacterial communities in undisturbed acidic forest soils were quite stable during seasonal change.     

Bacterial diversity in the bulk soil and rhizosphere fractions of Lolium perenne and Trifolium repens as revealed by PCR restriction analysis of 16S rDNA

The rhizosphere of Trifolium repens and Lolium perenne was divided into three fractions: the bulk soil, the soil adhering to the roots and the washed roots (rhizoplane and endorhizosphere). After isolation and purification of DNA from these fractions, 16S rDNA was amplified by PCR and cloned to obtain a collection of 16S rRNA genes representative of the bacterial communities of these three fractions. The genes were then characterized by PCR restriction analysis. Each different profile was used to define an operational taxonomic unit (OTU). The numbers of OTUs and the numbers of clones among these OTUs allowed to calculate a diversity index. The number of OTUs decreased as root proximity increased and a few OTUs became dominant, resulting in a lower diversity index. In the root fraction of T. repens, the restriction profile of the dominant OTU matched the theoretical profile of the 16S rRNA gene of Rhizobium leguminosarum. This study showed that plant roots create a selective environment for microbial populations.     

Microbial community composition but not diversity changes along succession in arctic sand dunes

The generality of increasing diversity of fungi and bacteria across arctic sand dune succession was tested. Microbial communities were examined by high‐throughput sequencing of 16S rRNA genes (bacteria) and internal transcribed spacer (ITS) regions (fungi). We studied four microbial compartments (inside leaf, inside root, rhizosphere and bulk soil) and characterized microbes associated with a single plant species (Deschampsia flexuosa) across two sand dune successional stages (early and late). Bacterial richness increased across succession in bulk soil and leaf endosphere. In contrast, soil fungal richness remained constant while root endosphere fungal richness increased across succession. There was, however, no significant difference in Shannon diversity indices between early and late successional stage in any compartment. There was a significant difference in the composition of microbial communities between early and late successional stage in all compartments, although the major microbial OTUs were shared between early and late successional stage. Co‐occurrence network analysis revealed successional stage‐specific microbial groups. There were more co‐occurring modules in early successional stage than in late stage. Altogether, these results emphasize that succession strongly affects distribution of microbial species, but not microbial diversity in arctic sand dune ecosystem and that fungi and bacteria may not follow the same successional trajectories.