Sample pooling obscures diversity patterns in intertidal ciliate community composition and structure

The aim of this study was to assess the effect of sample pooling on the portrayal of ciliate community structure and composition in intertidal sediment samples. Molecular ciliate community profiles were obtained from nine biological replicates distributed in three discrete sampling plots and from samples that were pooled prior to RNA extraction using terminal restriction fragment polymorphism (T-RFLP) analyses of SSU rRNA. Comparing the individual replicates of one sampling plot with each other, we found a differential variability among the individual biological replicates. T-RFLP profiles of pooled samples displayed a significantly different community composition compared with the cumulative individual biological replicate samples. We conclude that sample pooling obscures diversity patterns in ciliate and possibly also other microbial eukaryote studies. However, differences between pooled samples and replicates were less pronounced when community structure was analyzed. We found that the most abundant T-RFLP peaks were generally shared between biological replicates and pooled samples. Assuming that the most abundant taxa in an ecosystem under study are also the ones driving ecosystem processes, sample pooling may still be effective for the analyses of ecological key players.     

Negative Effects of Sample Pooling on PCR-Based Estimates of Soil Microbial Richness and Community Structure

In this study, we examined the effect of various pooling strategies on the characterization of soil microbial community composition and phylotype richness estimates. Automated ribosomal intergenic spacer analysis (ARISA) profiles were determined from soil samples that were (i) unpooled (extracted and amplified individually), (ii) pooled prior to PCR amplification, or (iii) pooled prior to DNA extraction. Regression analyses suggest that the less even the soil microbial community (i.e., low Shannon equitability, E_H), the greater was the impact of either pooling strategy on microbial detection (R² = 0.766). For example, at a tropical rainforest site, which had the most uneven fungal (E_H of 0.597) and bacterial communities (E_H of 0.822), the unpooled procedure detected an additional 67 fungal and 115 bacterial phylotypes relative to either of the pooled procedures. Phylotype rarity, resulting in missed detection upon pooling, differed between the fungal and bacterial communities. Fungi were typified by locally abundant but spatially rare phylotypes, and the bacteria were typified by locally rare but spatially ubiquitous phylotypes. As a result, pooling differentially influenced plot comparisons, leading to an increase in similarity for the bacterial community and a decrease in the fungal community. In conclusion, although pooling reduces sample numbers and variability, it could mask a significant portion of the detectable microbial community, particularly for fungi due to their higher spatial heterogeneity.Microbial communities in soils are extremely complex, with heterogeneity expressed on a wide variety of scales (6-9, 16). Therefore, soil sampling strategies typically combine multiple small samples, obtained from various locations within the area of interest, into a single homogenized sample that is then subsampled for analysis. Previous studies (5, 11, 15) have compared the sizes of the subsamples to best represent the microbial diversity in the pooled samples. Larger sample sizes are typically recommended for community profiling (5, 11, 15) because they can reduce variability in the subsample and appear to adequately capture the dominant members of the community (3, 11). Conversely, multiple small subsamples have been proposed to be better suited for identifying rare community members and estimating species richness (10-11). While previous studies have largely been conducted to determine the variability of the subsample—and, hence, its ability to represent the larger, homogenized sample—the impact of soil sample size and pooling to best represent the site of interest and its influence on plot comparisons has not been adequately explored. For example, “rare” species in the pooled, homogenized sample may arise from two different scenarios: (i) species are found in high abundance at fine scales but are heterogeneously spaced, and (ii) species are found in low abundance but are ubiquitously distributed. Furthermore, detection of rare species could be problematic with molecular approaches that rely on PCR for amplification and detection.Although molecular techniques can detect many microbial species missed by traditional culturing (20), they suffer from several potential biases (14, 17) that may limit successful PCR amplification and detection. For example, because PCR is a competitive process, species with a low relative abundance will be amplified to a lesser degree and may not reach detection threshold levels. This process is routinely utilized in competitive PCR to analyze starting template concentrations in mixed nucleic acid samples (17, 19). Furthermore, this effect would be seen by any process that could dilute rare phylotypes, such as pooling DNA extracts prior to amplification. Therefore, if the microbial community in the starting template is too complex, reducing the soil sample size will increase the likelihood that less abundant species are successfully amplified and detected.In this study, we analyzed the influence of three different sampling strategies on microbial community profiling using automated ribosomal intergenic spacer analysis (ARISA) and the following types of samples: (i) unpooled, (ii) pooled prior to PCR amplification, or (iii) pooled prior to DNA extraction. This sampling scheme was designed to test the effects of different common sampling strategies on microbial community profiles of samples containing equal soil volumes. Studies were conducted at three different field sites with various types of plant overstory complexity: an agricultural corn field, a ponderosa pine forest, and a tropical rainforest.     

Soil parent material is a key determinant of the bacterial community structure in arable soils

The bacterial community composition in soil and rhizosphere taken from arable field sites, differing in soil parent material and soil texture, was analyzed using terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes. Nine sandy to silty soils from North-East Germany could clearly be distinguished from each other, with a relatively low heterogeneity in the community structure within the field replicates. There was a relationship between the soil parent material, i.e. different glacial and aeolian sediments, and the clustering of the profiles from different sites. A site-specific grouping of T-RFLP profiles was also found for the rhizosphere samples of the same field sites that were planted with potatoes. The branching of the rhizosphere profiles corresponded partly with the soil parent material, whereas the effect of the plant genotype was negligible. Selected terminal restriction fragments differing in their relative abundance within the nine soils were analyzed based on the cloning of the 16S rRNA genes of one soil sample. A high phylogenetic diversity observed to include Acidobacteria, Betaproteobacteria, Bacteroidetes, Verrucomicrobia, and Gemmatimonadetes. The assignment of three out of the seven selected terminal restriction fragments to members of Acidobacteria suggested that this group seems to participate frequently in the shifting of community structures that result from soil property changes.     

Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques

Fungi fulfil a range of important ecological functions, yet current understanding of fungal biodiversity in soil is limited. Direct DNA extraction from soil, coupled with polymerase chain reaction amplification and community profiling techniques, has proved successful in investigations of bacterial ecology and shows great promise for elucidating the taxonomic and functional characteristics of soil fungal communities. These community profiling techniques include denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), single-strand conformation polymorphism (SSCP), terminal restriction fragment length polymorphism (T-RFLP), amplified rDNA restriction analysis (ARDRA), amplified ribosomal intergenic spacer analysis (ARISA) and cloning, and are generally coupled with DNA sequencing. The techniques and their potential limitations are discussed, along with recent advances that have been made possible through their application in soil fungal ecology. It is unlikely that a single approach will be universally applicable for assessing fungal diversity in all soils or circumstances. However, judicious selection of the methodology, keeping the experimental aims in mind, and the exploitation of emerging technologies will undoubtedly increase our understanding of soil fungal communities in the future.     

A novel strategy to extract specific phylogenetic sequence information from community T-RFLP

Cultivation-independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. Semi-automated terminal restriction fragment length polymorphism (T-RFLP) analyses yield high-resolution genetic profiles of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring. A serious limitation of T-RFLP analyses has been the inability to reliably affiliate observed terminal restriction fragments (T-RF) to phylogenetic groups. In the study presented here, we were able to overcome this limitation of T-RFLP. With a combination of adapter ligation, fragment size selection, and re-amplification with adapter site specific PCR, we were able to isolate a T-RF-fraction of a narrow size-range containing a T-RF that was significantly more abundant in heavy metal amended soils. Cloning the size-selected T-RF fraction allowed for the efficient isolation of clones containing this specific T-RF. Sequence determination and phylogenetic inference in RDP-II affiliated the sequence to unclassified cyanobacteria. Specific primer design and PCR amplification from bulk soil DNA allowed for independent confirmation of the results from bacterial T-RFLP and T-RF cloning. Our results show that specific T-RFs can be efficiently isolated and identified, and that the adapter ligation approach holds great potential for genetic profiling and for identification of community components of interest.     

Microbial DNA profiling by multiplex terminal restriction fragment length polymorphism for forensic comparison of soil and the influence of sample condition

Aims:  To evaluate: (i) the impact of air-drying on bacterial, archaeal and fungal soil DNA profiles and (ii) the potential use of multiplex-terminal restriction fragment length polymorphism (M-TRFLP) as a tool for forensic comparison of soil.
Methods and Results:  An M-TRFLP approach was used to profile bacterial, archaeal and fungal DNA profiles from five different soil sites. Air-drying soil significantly reduced the quantity of DNA but the number of operational taxanomic units (OTU) was unaffected. The impact of air-drying on soil DNA profiles was dependent on soil site and microbial primers. Fungal profiles were altered the least by air-drying. For prokaryotic profiles, air-drying altered the relative similarity/dissimilarity between soil sites. The M-TRFLP approach was more discriminatory compared with soil colour and single-taxa profiling, but did not significantly improve resolution between two similar soils.
Conclusions:  Of those tested, soil fungi were potentially the more robust target for application to soil forensic studies as they were altered less by air-drying and provided clear discrimination of soils from different sites. The M-TRFLP method demonstrated potential to achieve greater resolution, discriminating the soil sites based on both bacterial and fungal components.
Significance and Impact of the Study:  Soil DNA profiling has potential as a forensic tool, but sample condition and the appropriate selection of microbial target taxa must be considered.     

Testing Potential Effects of Maize Expressing the Bacillus thuringiensis Cry1Ab Endotoxin (Bt Maize) on Mycorrhizal Fungal Communities via DNA- and RNA-Based Pyrosequencing and Molecular Fingerprinting

The cultivation of genetically modified (GM) crops has increased significantly over the last decades. However, concerns have been raised that some GM traits may negatively affect beneficial soil biota, such as arbuscular mycorrhizal fungi (AMF), potentially leading to alterations in soil functioning. Here, we test two maize varieties expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) for their effects on soil AM fungal communities. We target both fungal DNA and RNA, which is new for AM fungi, and we use two strategies as an inclusive and robust way of detecting community differences: (i) 454 pyrosequencing using general fungal rRNA gene-directed primers and (ii) terminal restriction fragment length polymorphism (T-RFLP) profiling using AM fungus-specific markers. Potential GM-induced effects were compared to the normal natural variation of AM fungal communities across 15 different agricultural fields. AM fungi were found to be abundant in the experiment, accounting for 8% and 21% of total recovered DNA- and RNA-derived fungal sequences, respectively, after 104 days of plant growth. RNA- and DNA-based sequence analyses yielded most of the same AM fungal lineages. Our research yielded three major conclusions. First, no consistent differences were detected between AM fungal communities associated with GM plants and non-GM plants. Second, temporal variation in AMF community composition (between two measured time points) was bigger than GM trait-induced variation. Third, natural variation of AMF communities across 15 agricultural fields in The Netherlands, as well as within-field temporal variation, was much higher than GM-induced variation. In conclusion, we found no indication that Bt maize cultivation poses a risk for AMF.     

Technicalities and Glitches of Terminal Restriction Fragment Length Polymorphism (T-RFLP)

Terminal restriction fragment length polymorphism (T-RFLP) is a rapid, robust, inexpensive and simple tool for microbial community profiling. Methods used for DNA extraction, PCR amplification and digestion of amplified products have a considerable impact on the results of T-RFLP. Pitfalls of the method skew the similarity analysis and compromise its high throughput ability. Despite a high throughput method of data generation, data analysis is still in its infancy and needs more attention. Current article highlights the limitations of the methods used for data generation and analysis. It also provides an overview of the recent methodological developments in T-RFLP which will assist the readers in obtaining real and authentic profiles of the microbial communities under consideration while eluding the inherent biases and technical difficulties.     

Effects of site and plant species on rhizosphere community structure as revealed by molecular analysis of microbial guilds

The bacterial and fungal rhizosphere communities of strawberry (Fragaria ananassa Duch.) and oilseed rape (Brassica napus L.) were analysed using molecular fingerprints. We aimed to determine to what extent the structure of different microbial groups in the rhizosphere is influenced by plant species and sampling site. Total community DNA was extracted from bulk and rhizosphere soil taken from three sites in Germany in two consecutive years. Bacterial, fungal and group-specific (Alphaproteobacteria, Betaproteobacteria and Actinobacteria) primers were used to PCR-amplify 16S rRNA and 18S rRNA gene fragments from community DNA prior to denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial fingerprints of soil DNA revealed a high number of equally abundant faint bands, while rhizosphere fingerprints displayed a higher proportion of dominant bands and reduced richness, suggesting selection of bacterial populations in this environment. Plant specificity was detected in the rhizosphere by bacterial and group-specific DGGE profiles. Different bulk soil community fingerprints were revealed for each sampling site. The plant species was a determinant factor in shaping similar actinobacterial communities in the strawberry rhizosphere from different sites in both years. Higher heterogeneity of DGGE profiles within soil and rhizosphere replicates was observed for the fungi. Plant-specific composition of fungal communities in the rhizosphere could also be detected, but not in all cases. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Rostock site revealed that Streptomyces sp. and Rhizobium sp. were among the dominant ribotypes in the strawberry rhizosphere, while sequences from Arthrobacter sp. corresponded to dominant bands from oilseed rape bacterial fingerprints.     

Influence of elevated CO(2) on the fungal community in a coastal scrub oak forest soil investigated with terminal-restriction fragment length polymorphism analysis

Sixteen open-top chambers (diameter, 3.66 m) were established in a scrub oak habitat in central Florida where vegetation was removed in a planned burn prior to chamber installation. Eight control chambers have been continuously exposed to ambient air and eight have been continuously exposed to elevated CO(2) at twice-ambient concentration (approximately 700 ppm) for 5 years. Soil cores were collected from each chamber to examine the influence of elevated atmospheric CO(2) on the fungal community in different soil fractions. Each soil sample was physically fractionated into bulk soil, rhizosphere soil, and roots for separate analyses. Changes in relative fungal biomass were estimated by the ergosterol technique. In the bulk soil and root fractions, a significantly increased level of ergosterol was detected in the elevated CO(2) treatments relative to ambient controls. Fungal community composition was determined by terminal-restriction fragment length polymorphism (T-RFLP) analysis of the internal transcribed spacer (ITS) region. The specificities of different ITS primer sets were evaluated against plant and fungal species isolated from the experimental site. Changes in community composition were assessed by principal component analyses of T-RFLP profiles resolved by capillary electrophoresis. Fungal species richness, defined by the total number of terminal restriction fragments, was not significantly affected by either CO(2) treatment or soil fraction.     

An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics

A systematic evaluation of the value and potential of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure has been undertaken. The reproducibility and robustness of the method has been assessed using environmental DNA samples isolated directly from PCB-polluted or pristine soil, and subsequent polymerase chain reaction (PCR) amplification of total community 16S rDNA. An initial investigation to assess the variability both within and between different polyacrylamide gel electrophoresis (PAGE) runs showed that almost identical community profiles were consistently produced from the same sample. Similarly, very little variability was observed as a result of variation between replicate restriction digestions, PCR amplifications or between replicate DNA isolations. Decreasing concentrations of template DNA produced a decline in both the complexity and the intensity of fragments present in the community profile, with no additional fragments detected in the higher dilutions that were not already present when more original template DNA was used. Reducing the number of cycles of PCR produced similar results. The greatest variation between profiles generated from the same DNA sample was produced using different Taq DNA polymerases, while lower levels of variability were found between PCR products that had been produced using different annealing temperatures. Incomplete digestion by the restriction enzyme may, as a result of the generation of partially digested fragments, lead to an overestimation of the overall diversity within a community. The results obtained indicate that, once standardized, T-RFLP analysis is a highly reproducible and robust technique that yields high-quality fingerprints consisting of fragments of precise sizes, which, in principle, could be phylogenetically assigned, once an appropriate database is constructed.     

Biases for detecting arbuscular mycorrhizal fungal mixture by terminal restriction fragment length polymorphism (T-RFLP)

Terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified ribosomal RNA genes is used for profiling microbial communities and sometimes for species richness and relative abundance estimation in environmental samples. However, the T-RFLP fingerprint may be subject to biases during the procedure, influencing the detection of real community structures in the environment. To investigate possible sources of T-RFLP bias, 18S rRNA gene clones derived from two arbuscular mycorrhizal fungal sequences were combined in simple pairwise mixes to assess the effects of polymerase chain reaction cycle number, plant genomic DNA purification method and varying template ratio on the template-to-product ratio as measured by relative T-RF peak area. Varying cycle numbers indicated that amplification was still in the exponential phase at the cycle numbers lower than 18, so these small cycle numbers were used for the comparison of template-to-product quantities. Relative abundance estimated from T-RF peak ratios varied with different purification procedures, but the best results, closest to input ratios, were obtained by using phenol–chloroform purification. The presence of an excess of unpurified non-target plant genomic DNA generated a bias towards lower or overestimation of relative abundance. We conclude that a low number of amplification cycles and stringent DNA purification are necessary for accurate mixed sample analysis by T-RFLP.     

Bacterial communities in Arctic fjelds of Finnish Lapland are stable but highly pH-dependent

The seasonal and spatial variations of microbial communities in Arctic fjelds of Finnish Lapland were studied. Phospholipid fatty acid analysis (PLFA) and terminal restriction fragment analysis (T-RFLP) of amplified 16S rRNA genes were used to assess the effect of soil conditions and vegetation on microbial community structures along different altitudes of two fjelds, Saana and Jehkas. Terminal restriction fragments were additionally analysed from c. 160 cloned sequences and isolated bacterial strains and matched with those of soil DNA samples. T-RFLP and PLFA analyses indicated relatively similar microbial communities at various altitudes and under different vegetation of the two fjelds. However, soil pH had a major influence on microbial community composition. Members of the phylum Acidobacteria dominated especially in the low pH soils (pH 4.6-5.2), but above pH 5.5, the relative amount of terminal restriction fragments corresponding to acidobacterial clones was substantially lower. Both T-RFLP and PLFA analysis indicated stable microbial communities as the DNA and fatty acid profiles were similar in spring and late summer samples sampled over 3 years. These results indicate that differences in microbial community composition could be explained primarily by variation in the bedrock materials that cause variation in the soil pH.     

Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants

The aim of this study was to examine whether the terminal restriction fragment length polymorphism (T-RFLP) analysis represents an appropriate technique for monitoring highly diverse soil bacterial communities, i.e. to assess spatial and/or temporal effects on bacterial community structure. The T-RFLP method, a recently described fingerprinting technique, is based on terminal restriction fragment length polymorphisms between distinct small-subunit rRNA gene sequence types. This technique permits an automated quantification of the fluorescence signal intensities of the individual terminal restriction fragments (T-RFs) in a given community fingerprint pattern. The indigenous bacterial communities of three soil plots located within an agricultural field of 110 m(2) were compared. The first site was planted with non-transgenic potato plants, while the other two were planted with transgenic GUS and Barnase/Barstar potato plants, respectively. Once prior to planting and three times after planting, seven parallel samples were taken from each of the three soil plots. The T-RFLP analysis resulted in very complex but highly reproducible community fingerprint patterns. The percentage abundance values of defined T-RFs were calculated for the seven parallel samples of the respective soil plot. A multivariate analysis of variance was used to test T-RFLP data sets for significant differences. The statistical treatments clearly revealed spatial and temporal effects, as well as spacextime interaction effects, on the structural composition of the bacterial communities. T-RFs which showed the highest correlations to the discriminant factors were not those T-RFs which showed the largest single variations between the seven-sample means of individual plots. In summary, the T-RFLP technique, although a polymerase chain reaction-based method, proved to be a suitable technique for monitoring highly diverse soil microbial communities for changes over space and/or time.     

Cultivation-Independent Analysis of Fungal Genotypes in Soil by Using Simple Sequence Repeat Markers

Cultivation-independent analyses of fungi are used for community profiling as well as identification of specific strains in environmental samples. The objective of the present study was to adapt genotyping based on simple sequence repeat (SSR) marker detection for use in cultivation-independent monitoring of fungal species or strains in bulk soil DNA. As a model system, a fungal biocontrol agent (BCA) based on Beauveria brongniartii, for which six SSR markers have been developed, was used. Species specificity of SSR detection was verified with 15 fungal species. Real-time PCR was used to adjust for different detection sensitivities of the six SSR markers as well as for different template quantities. The limit for reliable detection per PCR assay was below 2 pg target DNA, corresponding to an estimated 45 genome copies of B. brongniartii. The cultivation-independent approach was compared to cultivation-dependent SSR analysis with soil samples from a B. brongniartii BCA-treated field plot. Results of the cultivation-independent method were consistent with cultivation-dependent genotyping and allowed for unambiguous identification and differentiation of the applied as well as indigenous strains in the samples. Due to the larger quantities of soil used for cultivation-dependent analysis, its sensitivity was higher, but cultivation-independent SSR genotyping was much faster. Therefore, cultivation-independent monitoring of B. brongniartii based on multiple SSR markers represents a rapid and strain-specific approach. This strategy may also be applicable to other fungal species or strains for which SSR markers have been developed.     

内蒙古草原典型植物对土壤微生物群落的影响

为了分析内蒙古草原不同植物物种对土壤微生物群落的影响, 采用实时荧光定量PCR (real-time PCR)以及末端限制性片段长度多态性分析(terminal restriction fragment length polymorphism, T-RFLP)等分子生物学技术, 测定了退化-恢复样地上几种典型植物的根际土壤和非根际土壤中细菌和真菌的数量及群落结构。结果表明, 不同植物物种对根际和非根际细菌及根际真菌数量均有显著影响。根际土壤中的细菌和真菌数量普遍高于非根际土壤, 尤其以真菌更为明显。对T-RFLP数据进行多响应置换过程(multi-response permutation procedures, MRPP)分析和主成分分析(principal component analysis, PCA), 结果表明, 大多数物种的根际细菌及真菌的群落结构与非根际有明显差异, 并且所有物种的真菌群落可以按根际和非根际明显分为两大类群。此外, 细菌和真菌群落结构在一定程度上存在按物种聚类的现象, 以细菌较为明显。这些结果揭示了不同植物对土壤微生物群落的影响特征, 对理解内蒙古草原地区退化及恢复过程中植被演替引起的土壤性质和功能的变化有一定的帮助。