Unveiling fungal zooflagellates as members of freshwater picoeukaryotes: evidence from a molecular diversity study in a deep meromictic lake

This study presents an original 18S rRNA PCR survey of the freshwater picoeukaryote community, and was designed to detect unidentified heterotrophic picoflagellates (size range 0.6-5 microm) which are prevalent throughout the year within the heterotrophic flagellate assemblage in Lake Pavin. Four clone libraries were constructed from samples collected in two contrasting zones in the lake. Computerized statistic tools have suggested that sequence retrieval was representative of the in situ picoplankton diversity. The two sampling zones exhibited similar diversity patterns but shared only about 5% of the operational taxonomic units (OTUs). Phylogenetic analysis clustered our sequences into three taxonomic groups: Alveolates (30% of OTUs), Fungi (23%) and Cercozoa (19%). Fungi thus substantially contributed to the detected diversity, as was additionally supported by direct microscopic observations of fungal zoospores and sporangia. A large fraction of the sequences belonged to parasites, including Alveolate sequences affiliated to the genus Perkinsus known as zooparasites, and chytrids that include host-specific parasitic fungi of various freshwater phytoplankton species, primarily diatoms. Phylogenetic analysis revealed five novel clades that probably include typical freshwater environmental sequences. Overall, from the unsuspected fungal diversity unveiled, we think that fungal zooflagellates have been misidentified as phagotrophic nanoflagellates in previous studies. This is in agreement with a recent experimental demonstration that zoospore-producing fungi and parasitic activity may play an important role in aquatic food webs.     

Only a Few Fungal Species Dominate Highly Diverse Mycofloras Associated with the Common Reed

Plants are naturally colonized by many fungal species that produce effects ranging from beneficial to pathogenic. However, how many of these fungi are linked with a single host plant has not been determined. Furthermore, the composition of plant-associated fungal communities has not been rigorously determined. We investigated these essential issues by employing the perennial wetland reed Phragmites australis as a model. DNA extracted from roots, rhizomes, stems, and leaves was used for amplification and cloning of internal transcribed spacer rRNA gene fragments originating from reed-associated fungi. A total of 1,991 clones from 15 clone libraries were differentiated by restriction fragment length polymorphism analyses into 345 operational taxonomical units (OTUs). Nonparametric estimators for total richness (Chao1 and ACE) and also a parametric log normal model predicted a total of about 750 OTUs if the libraries were infinite. Sixty-two percent of the OTUs sequenced were novel at a threshold of 3%. Several of these OTUs represented undocumented fungal species, which also included higher taxonomic levels. In spite of the high diversity of the OTUs, the mycofloras of vegetative organs were dominated by just a few typical fungi, which suggested that competition and niche differentiation influence the composition of plant-associated fungal communities. This suggestion was independently supported by the results of nested PCR assays specifically monitoring two OTUs over 3 years, which revealed significant preferences for host habitat and host organ.     

Could nested PCR be applicable for the study of microbial diversity?

PCR-based methods for rRNA gene analysis have been widely used to study diversity of microbiology. However, the analysis would be difficult when the DNA content in samples is too low to be amplified by conventional PCR. Nested PCR comes up with the advantage of higher sensitivity. It can detect target DNA at several-fold lower concentrations than conventional PCR. However, the amplification bias and factors that potentially affect measurement of sample diversity associated with nested PCR method has received little attention. Here, nested PCR was compared to reconditioning PCR which is based on conventional PCR and it would reduce the formation of heteroduplex. We investigated the use of both nested and reconditioning PCR methods to construct clone libraries of 16S rRNA genes from four swimming pool water samples. Abundances of OTUs (operational taxonomic units) were correlated between the libraries (r ² = 0.88, P < 0.0005), and some OTUs had equivalent abundances in the two libraries using the Chi-square test. Differences in taxonomic groups, as well as diversity and richness estimators, were compared by paired t-test and the Wilcoxon test, respectively. There were no significant differences between clone libraries using these two PCR methods. The results of ∫-Libshuff analysis suggested that nested PCR have no particular biases in revealing OTU diversity of a bacterial community. Thus, nested PCR produce congruent pictures with reconditioning PCR in the microbial community analysis.     

Sanger和Pyrosequencing测序法分析健康成人口腔菌群

目的对比Sanger和Pyrosequencing测序法分析健康人口腔菌群组成。方法收集6例健康成人唾液、舌背、黏膜、龈上及龈下菌斑并构建16SrRNA基因文库,分别用Sanger和Pyrosequencing测序法分析。结果 Sanger测序所得已知的序列有5,794条(占6,535总序列数88.7%)、75个属,396个序列划分操作分类单元(operational taxonomic units,OTUs,占总OTUs的61.4%)。Pyrosequencing测序所得已知的序列有10,771条(占11,103总序列数97.0%)、66个属,322个OTUs(占总OTUs的68.0%)。Sanger和Pyrosequencing测序法所得口腔菌群在门、属的水平分布趋势基本一致,但在种的水平分布差异显著。Sanger和Pyrosequencing测序法构建的口腔菌群文库均匀度值分别为0.016和0.007,说明Pyrosequencing分析口腔菌群物种数量分布比Sanger测序方法的文库均匀性稍差,但优势种更显著。结论 Pyrosequencing测序时所构建基因文库能代表口腔菌群的多样性且经济、省时,可以应用于口腔细菌物种的分析。     

Diversity and quantity of ammonia-oxidizing Archaea and Bacteria in sediment of the Pearl River Estuary, China

The diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the sediment of the Pearl River Estuary were investigated by cloning and quantitative real-time polymerase chain reaction (qPCR). From one sediment sample S16, 36 AOA OTUs (3% cutoff) were obtained from three clone libraries constructed using three primer sets for amoA gene. Among the 36 OTUs, six were shared by all three clone libraries, two appeared in two clone libraries, and the other 28 were only recovered in one of the libraries. For AOB, only seven OTUs (based on 16S rRNA gene) and eight OTUs (based on amoA gene) were obtained, showing lower diversity than AOA. The qPCR results revealed that AOA amoA gene copy numbers ranged from 9.6 × 10⁶ to 5.1 × 10⁷ copies per gram of sediment and AOB amoA gene ranged from 9.5 × 10⁴ to 6.2 × 10⁵ copies per gram of sediment, indicating that the dominant ammonia-oxidizing microorganisms in the sediment of the Pearl River Estuary were AOA. The terminal restriction fragment length polymorphism results showed that the relative abundance of AOB species in the sediment samples of different salinity were significantly different, indicating that salinity might be a key factor shaping the AOB community composition.     

High consistency between replicate 454 pyrosequencing analyses of ectomycorrhizal plant root samples

In this methodological study, we compare 454 sequencing and a conventional cloning and Sanger sequencing approach in their ability to characterize fungal communities PCR amplified from four root systems of the ectomycorrhizal plant Bistorta vivipara. To examine variation introduced by stochastic processes during the laboratory work, we replicated all analyses using two independently obtained DNA extractions from the same root systems. The ITS1 region was used as DNA barcode and the sequences were clustered into OTUs as proxies for species using single linkage clustering (BLASTClust) and 97% sequence similarity cut-off. A relatively low overlap in fungal OTUs was observed between the 454 and the clone library datasets — even among the most abundant OTUs. In a non-metric multidimensional scaling analysis, the samples grouped more according to methodology compared to plant. Some OTUs frequently detected by 454, most notably those OTUs with taxonomic affinity to Glomales, were not detected in the Sanger dataset. Likewise, a few OTUs, including Cenococcum sp., only appeared in the clone libraries. Surprisingly, we observed a significant relationship between GC/AT content of the OTUs and their proportional abundances in the 454 versus the clone library datasets. Reassuringly, a very good consistency in OTU recovery was observed between replicate runs of both sequencing methods. This indicates that stochastic processes had little impact when applying the same sequencing technique on replicate samples.     

Phylogenetic diversity of bacterial communities in bovine rumen as affected by diets and microenvironments

Phylogenetic analysis was conducted to examine ruminal bacteria in two ruminal fractions (adherent fraction vs. liquid fraction) collected from cattle fed with two different diets: forage alone vs. forage plus concentrate. One hundred forty-four 16S rRNA gene (rrs) sequences were obtained from clone libraries constructed from the four samples. These rrs sequences were assigned to 116 different operational taxonomic units (OTUs) defined at 0.03 phylogenetic distance. Most of these OTUs could not be assigned to any known genus. The phylum Firmicutes was represented by approximately 70% of all the sequences. By comparing to the OTUs already documented in the rumen, 52 new OTUs were identified. UniFrac, SONS, and denaturing gradient gel electrophoresis analyses revealed difference in diversity between the two fractions and between the two diets. This study showed that rrs sequences recovered from small clone libraries can still help identify novel species-level OTUs.     

Only a few fungal species dominate highly diverse mycofloras associated with the common reed

Plants are naturally colonized by many fungal species that produce effects ranging from beneficial to pathogenic. However, how many of these fungi are linked with a single host plant has not been determined. Furthermore, the composition of plant-associated fungal communities has not been rigorously determined. We investigated these essential issues by employing the perennial wetland reed Phragmites australis as a model. DNA extracted from roots, rhizomes, stems, and leaves was used for amplification and cloning of internal transcribed spacer rRNA gene fragments originating from reed-associated fungi. A total of 1,991 clones from 15 clone libraries were differentiated by restriction fragment length polymorphism analyses into 345 operational taxonomical units (OTUs). Nonparametric estimators for total richness (Chao1 and ACE) and also a parametric log normal model predicted a total of about 750 OTUs if the libraries were infinite. Sixty-two percent of the OTUs sequenced were novel at a threshold of 3%. Several of these OTUs represented undocumented fungal species, which also included higher taxonomic levels. In spite of the high diversity of the OTUs, the mycofloras of vegetative organs were dominated by just a few typical fungi, which suggested that competition and niche differentiation influence the composition of plant-associated fungal communities. This suggestion was independently supported by the results of nested PCR assays specifically monitoring two OTUs over 3 years, which revealed significant preferences for host habitat and host organ.     

PhylOTU: a high-throughput procedure quantifies microbial community diversity and resolves novel taxa from metagenomic data

Microbial diversity is typically characterized by clustering ribosomal RNA (SSU-rRNA) sequences into operational taxonomic units (OTUs). Targeted sequencing of environmental SSU-rRNA markers via PCR may fail to detect OTUs due to biases in priming and amplification. Analysis of shotgun sequenced environmental DNA, known as metagenomics, avoids amplification bias but generates fragmentary, non-overlapping sequence reads that cannot be clustered by existing OTU-finding methods. To circumvent these limitations, we developed PhylOTU, a computational workflow that identifies OTUs from metagenomic SSU-rRNA sequence data through the use of phylogenetic principles and probabilistic sequence profiles. Using simulated metagenomic data, we quantified the accuracy with which PhylOTU clusters reads into OTUs. Comparisons of PCR and shotgun sequenced SSU-rRNA markers derived from the global open ocean revealed that while PCR libraries identify more OTUs per sequenced residue, metagenomic libraries recover a greater taxonomic diversity of OTUs. In addition, we discover novel species, genera and families in the metagenomic libraries, including OTUs from phyla missed by analysis of PCR sequences. Taken together, these results suggest that PhylOTU enables characterization of part of the biosphere currently hidden from PCR-based surveys of diversity?     

利用小亚基核糖体RNA 技术分析温室黄瓜近根土壤古菌和真菌多样性

土壤古菌和真菌在温室生态系统是仅次于细菌的微生物,具有类似于细菌的重要生态功能。通过构建古菌16S rRNA和真菌18S rRNA基因克隆文库,分析温室黄瓜近根土壤古菌和真菌群落结构组成,为开发利用温室这一特殊的生态环境中丰富的微生物资源以及理解微生物与植物间的互作提供参考依据。采用研磨-冻融-溶菌酶-蛋白酶K-SDS热处理以及CTAB处理等理化方法,提取和纯化微生物总DNA,构建古菌16S rRNA和真菌18S rRNA基因克隆文库。利用DOTUR软件将古菌和真菌序列按照相似性97％的标准分成若干个可操作分类单元 (OTUs)。土壤古菌克隆文库主要包括泉古菌门和未分类的古菌两大类,并有少部分广域古菌类群,所有泉古菌均属于热变形菌纲,共45个OTUs;真菌克隆文库包括真菌门的大多数亚门真菌,共24个OTUs,未发现担子菌亚门真菌。古菌多样性比较丰富,且发现少量的广域古菌 (甲烷菌),这一情况可能与温室长期高温高湿,高有机质含量,土壤处于缺氧环境有关;土壤真菌的优势种群为子囊菌,占到土壤真菌的80％以上,这可能与绝大多数植物真菌性病害属于土传病害,通过菌丝体、菌核或子囊壳在土壤病残体中越冬有一定的关系。     

A universal cloning vector using vaccinia topoisomerase I

Vaccinia DNA topoisomerase I (TOPO) charged vectors with a sticky T are routinely used to clone polymerase chain reaction (PCR) products with an extra A at their 3′ end (TOPO TA Cloning from Invitrogen). TOPO charged blunt vectors are used to clone blunt end PCR products (TOPO Blunt Cloning). Here, we demonstrate that both TOPO TA vectors and TOPO Blunt vectors can be used to clone PCR products with either a blunt end or an extra A at the 3′ end. We further demonstrate that these vectors can be used to clone sticky end DNA generated with restriction enzymes. In summary, these TOPO vectors can be used as universal cloning vectors.     

Characterization of fungal communities associated with the bark beetle Ips typographus varies depending on detection method, location, and beetle population levels

The Eurasian spruce bark beetle Ips typographus and their fungal associates can cause severe damage to Norway spruce forests. In this paper, by using both molecular and cultural methods, we compared fungal assemblages on bark beetles from different locations, characterized by different beetle population levels. Ips typographus was trapped in the western Alps in two outbreak and in two control areas. Sequencing of clone libraries of Internal Transcribed Spacer (ITS) identified 31 fungal Operational Taxonomic Units (OTUs), while fungal isolations yielded 55 OTUs. Only three OTUs were detected by both molecular and cultural methods indicating that both methods are necessary to adequately describe fungal richness. Fungal assemblages on insects from these four and from an additional 12 study sites differed among stands in response to varying ecological conditions and to the limited spreading ability of I. typographus. Ophiostomatoid fungi showed higher diversity in outbreak areas; the pathogenic Ophiostoma polonicum was relatively uncommon, while O. bicolor was the most abundant species. This result was not unexpected, as insects were trapped not at the peak but at the end of the outbreaks and supports the hypothesis of a temporal succession among Ophiostoma species. Ubiquitous endophytes of trees or common airborne fungi were present both in outbreak and in control areas. Wood decaying basidiomycetes were almost never detected on beetles. Yeasts were detected only by molecular analysis, and the OTUs detected matched those reported elsewhere in Europe and in the world, suggesting a very long association between some yeasts and bark beetles.     

Capturing diversity of marine heterotrophic protists: one cell at a time

Recent applications of culture-independent, molecular methods have revealed unexpectedly high diversity in a variety of functional and phylogenetic groups of microorganisms in the ocean. However, none of the existing research tools are free from significant limitations, such as PCR and cloning biases, low phylogenetic resolution and others. Here, we employed novel, single-cell sequencing techniques to assess the composition of small (<10 μm diameter), heterotrophic protists from the Gulf of Maine. Single cells were isolated by flow cytometry, their genomes amplified, and 18S rRNA marker genes were amplified and sequenced. We compared the results to traditional environmental PCR cloning of sorted cells. The diversity of heterotrophic protists was significantly higher in the library of single amplified genomes (SAGs) than in environmental PCR clone libraries of the 18S rRNA gene, obtained from the same coastal sample. Libraries of SAGs, but not clones contained several recently discovered, uncultured groups, including picobiliphytes and novel marine stramenopiles. Clone, but not SAG, libraries contained several large clusters of identical and nearly identical sequences of Dinophyceae, Cercozoa and Stramenopiles. Similar results were obtained using two alternative primer sets, suggesting that PCR biases may not be the only explanation for the observed patterns. Instead, differences in the number of 18S rRNA gene copies among the various protist taxa probably had a significant role in determining the PCR clone composition. These results show that single-cell sequencing has the potential to more accurately assess protistan community composition than previously established methods. In addition, the creation of SAG libraries opens opportunities for the analysis of multiple genes or entire genomes of the uncultured protist groups.     

Probe-based negative selection for underrepresented phylotypes in large environmental clone libraries

Studies based on cloning and sequencing to investigate microbial diversity in a vast range of samples has become widespread in recent years. Results have revealed immense microbial diversity in many different environments, but also dominance of a few sequence types in the constructed clone libraries. Here we describe a method to enrich the clone libraries by avoiding sequencing of known, abundant sequence types, instead focusing on novel, rare ones. The protocol is based on gridding the PCR products from clone libraries on membranes and hybridisation of species-specific probes. Clones that do not give positive hybridisation results are sequenced. This method was used for fungal clone libraries from compost samples. Altogether 1536 clones were gridded and six probes used. From these clones, 59% hybridised with a probe, and therefore, only 41% of the clones were sequenced. In addition, 384 samples were sequenced to verify the hybridisation results. The numbers of false-negative (5.2%) and false-positive (3.9%) hybridisations were low. This method provides a mean of lowering the costs of sequencing projects and speeding up the process of characterising microbial diversity in environmental samples. The method is especially suitable for samples with a few dominating sequence types.     

喹啉与吲哚驯化的反硝化反应器的微生物群落结构分析比较

[目的]本研究旨在比较分析分别以喹啉和吲哚为底物,在相同条件下驯化的两个反硝化生物反应器的微生物群落结构.[方法]采用相同的种子污泥和相同的驯化条件,经过大约6周的驯化后,两个反应器均达到稳定而高效的污染物去除能力,通过16S rDNA克隆文库技术对两个反应器的微生物群落结构进行研究.[结果]研究发现,微生物群落结构表现出很大的差异.喹啉驯化的群落中所有的OTU都属于Betaproteobacteria,而吲哚驯化的群落中Betaproteobacteria占56.3%,吲哚驯化的群落具有更高的多样性.两个群落的优势OTU也不同,喹啉驯化群落中Thauera及其它Rhodocyclaceae科的微生物占整个群落的73%,而吲哚驯化群落中优势OTU为Comamonadaceae科、Alcaligenaceae科和Rhodocyclaceae科等类型的微生物,其中Comamonadaceae科的一个OTU占整个群落的28.7%.[结论]不同的驯化底物对微生物群落的组成具有较强的选择作用.首次报道并比较了可高效降解喹啉和吲哚的反硝化生物反应器的微生物群落结构.     

Comparative analysis of eukaryotic marine microbial assemblages from 18S rRNA gene and gene transcript clone libraries by using different methods of extraction

Eukaryotic marine microbes play pivotal roles in biogeochemical nutrient cycling and ecosystem function, but studies that focus on the protistan biogeography and genetic diversity lag-behind studies of other microbes. 18S rRNA PCR amplification and clone library sequencing are commonly used to assess diversity that is culture independent. However, molecular methods are not without potential biases and artifacts. In this study, we compare the community composition of clone libraries generated from the same water sample collected at the San Pedro Ocean Time Series (SPOTs) station in the northwest Pacific Ocean. Community composition was assessed using different cell lysis methods (chemical and mechanical) and the extraction of different nucleic acids (DNA and RNA reverse transcribed to cDNA) to build Sanger ABI clone libraries. We describe specific biases for ecologically important phylogenetic groups resulting from differences in nucleic acid extraction methods that will inform future designs of eukaryotic diversity studies, regardless of the target sequencing platform planned.     

长庆油田延9低渗透油藏微生物群落研究

目的:研究长庆油田延9低渗透油藏微生物群落,为实施微生物提高原油采收率提供指导和依据。方法:长庆油田延9油藏三口不同油井(柳28-46、柳28-47和柳27-45)的油水样品建立16S rDNA克隆文库进行研究。结果:构建了柳28-46、柳28-47和柳27-45油井样品的微生物基因克隆文库,其分类操作单元(OUT)数分别为21、20和20个;序列分析比对表明,3口井的共同的优势微生物菌群为铜绿假单胞菌(Pseudomonas aeruginos),分别占各文库的32.8%、32%和42.9%,它是最常见最主要的采油功能菌之一。此外硫酸盐还原菌(SRB)和铁细菌也处于优势地位,它们是原油开采中的有害菌。结论:延9低渗透油藏微生物群落和其潜在功能的分析为开展微生物提高石油采收率应用提供了良好的基础资料。     

Diversity measures in environmental sequences are highly dependent on alignment quality--data from ITS and new LSU primers targeting basidiomycetes

The ribosomal DNA comprised of the ITS1-5.8S-ITS2 regions is widely used as a fungal marker in molecular ecology and systematics but cannot be aligned with confidence across genetically distant taxa. In order to study the diversity of Agaricomycotina in forest soils, we designed primers targeting the more alignable 28S (LSU) gene, which should be more useful for phylogenetic analyses of the detected taxa. This paper compares the performance of the established ITS1F/4B primer pair, which targets basidiomycetes, to that of two new pairs. Key factors in the comparison were the diversity covered, off-target amplification, rarefaction at different Operational Taxonomic Unit (OTU) cutoff levels, sensitivity of the method used to process the alignment to missing data and insecure positional homology, and the congruence of monophyletic clades with OTU assignments and BLAST-derived OTU names. The ITS primer pair yielded no off-target amplification but also exhibited the least fidelity to the expected phylogenetic groups. The LSU primers give complementary pictures of diversity, but were more sensitive to modifications of the alignment such as the removal of difficult-to align stretches. The LSU primers also yielded greater numbers of singletons but also had a greater tendency to produce OTUs containing sequences from a wider variety of species as judged by BLAST similarity. We introduced some new parameters to describe alignment heterogeneity based on Shannon entropy and the extent and contents of the OTUs in a phylogenetic tree space. Our results suggest that ITS should not be used when calculating phylogenetic trees from genetically distant sequences obtained from environmental DNA extractions and that it is inadvisable to define OTUs on the basis of very heterogeneous alignments.