European Journal of Soil Biology 47 (2011) 77e87Contents lists available at ScienceDirect European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi Review A review of molecular methods to study the microbiota of soil and the mycosphere J.D. van Elsas*, F.G.H. Boersma Department of Microbial Ecology, CEES, University of Groningen, Kerklaan 30, 9750 RA Haren, Netherlands a r t i c l e i n f o a b s t r a c t Article history: Received 28 July 2010 Received in revised form 26 November 2010 Accepted 30 November 2010 Available online 15 December 2010 Handling editor: Bryan Griffiths The availability of novel and advanced molecular methods based on soil nucleic acids has revolutionized our studies of the microbiota of soil. In particular, our understanding of the daunting diversity of soil microbes has grown to maturity, opening up a new box of challenging research questions about microbial functioning and interactions. We here review recent developments in, as well as the stateof-the-art of, the molecular methods applied to soil, and discuss a few salient cases in which they have enhanced our understanding of the soil microbiota and its functioning. In particular, we place a focus on the interface between soil fungal hyphae and the corresponding non-fungal-affected soil, i.e., the mycosphere. This selective environment may reduce the diversity of its inhabitants, allowing an improved picture of their ecology and functioning via molecular techniques. We present arguments for the contention that, to investigate testable hypotheses, a polyphasic approach is needed, in which work on the basis of molecular approaches such as metagenomics and metatranscriptomics is coupled to that based on culturable organisms. Thus, advances in our understanding of local functioning and adaptation of bacterial mycosphere inhabitants will be fostered by combined metagenomics/metatranscriptomics and cultivation-based approaches. Ó 2010 Elsevier Masson SAS. All rights reserved. Keywords: Molecular methods Soil microbiota Mycosphere 1. Introduction The analysis of microbial populations in natural habitats such as soil is one of the cornerstones of current research on the functioning of natural ecosystems. In traditional soil microbiological approaches, data on soil microorganisms have been obtained by analyzing material derived from microbial growth, i.e., cells in liquid cultures or colonies obtained by plating. Methods derived from microbiology, cellular biochemistry, molecular biology (DNAor RNA-based) and physiology have traditionally been used with such material. However, such methods have often met with strong limitations, the reason being that only a small fraction of the microbiota in soil can be accessed on the basis of cultivation. This phenomenon has been coined the Great Plate Count Anomaly [1]. Researchers thus soon realized that the only sensible way to understand the complex soil microbial community was by developing direct molecular assessments, for which pre-extraction of cellular macromolecules like DNA and/or RNA was a prerequisite. In the light of the astounding development of analytical methods in molecular biology ever since the 1980-ies, exciting opportunities * Corresponding author. E-mail address:
[email protected] (J.D. van Elsas). 1164-5563/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2010.11.010 for analyses were offered if DNA and/or ribosomal or messenger RNA could be efficiently extracted from soil and subsequently analyzed [2,3]. The molecularly-based methods specifically enable to produce “snapshots” of the molecular make-up of whole complex soil microbial communities, as well as of specific microorganisms and genes therein. Although the term had not been coined at that time, this early stage of soil molecular microbiology may be rightly called the era of early metagenomics or “Protometagenomics”. The Molecular Microbial Ecology Manual (editions I and II) bears testimony of the ultrafast developments in this area over the past one to one-and-a-half decade [4,5]. A now almost traditional way of performing a molecular assessment of soil microbial communities following cultivation consists of colony hybridization, using suitable probes as proxies for the identification of the colonies e grown on isolation plates e that are examined. Early analyses of colony material have been based on this method [6], which allowed the investigators to pinpoint the presence of particular genes in their cultured organisms. The method was later followed by polymerase chain reaction (PCR)based assessments of colony material (colony PCR) directly from isolation plates. Such cultivation-based molecular analyses allowed the description of the population dynamics of specific culturable bacteria in soil settings. However, they were inherently limited in their scope due to the general unculturability of a majority of soil rigorous standardization is required in comparative work in soil. In contrast. As the hyphae.20. The commercial extraction kits for DNA. it is extremely important to consider and apply standardized sampling procedures of the mycosphere. still seem to limit the scope of the investigator to the (micro)habitat that is actually accessed.D. to keep the putative biases similar across samples or treatments. shaking the loose soil from the foot. the mycorrhizosphere e which includes influences from plant roots e can be recognized [11. This review will examine the molecular methods that are currently applied to soil and mycosphere systems. The mycosphere does not a priori pose major problems for DNA/RNA extractions using common procedures.21]. soil ectomycorrhizae. we address the ins and outs of the application of molecular methods to the mycosphere. van Elsas. culminating in the fact that current nucleic acid extraction protocols are almost all (commercial) kit-based [8]. Ideally. Such methods commonly yielded both DNA and RNA released from soil microbial communities.. finally. the reason being the greater stability of DNA upon extraction.14. molecular analyses applied specifically to such samples in comparison to those of the bulk soil will shed light on the specific molecular features (phylogenetically and/or functionally) of mycosphere inhabitants. The difficulty is also reflected in the definition of the mycosphere. relevant analytical procedures are examined with respect to their power to detect. Therefore.16]. advanced methods for the analysis of the diversity and community structures of soil-. On the other hand. Since their inception. the examination of organisms that are able to give a growth response is still highly crucial in many studies. It encompasses the soil zone around the hyphae of a range of soil fungi. and it is therefore not well-defined.and mycosphere-associated bacterial communities are the focus [10. several non-kit based protocols are still in use [8]. their ready readsorption and. a main challenge in studying this interface environment by molecular and/or cultivation means lies in the ability of the sampling procedure to dissect out the relevant portion of the soil that is impacted by fungal hyphae. the enzymatic (soft) cell lysis methods [2. arbuscular mycorrhizae and/or saprotrophic fungi [13]. and accepted sampling procedures based on statistical considerations are in place. plant. However. which is per definition extraradical. fingerprint.18]. In particular. Thus. incomplete and biased cell lyses. Key issues in soil nucleic acid extraction are the efficiencies of the release of microbial cells from soil particles and the subsequent lysis of the former. for specific purposes. to sensibly address the microbial communities of the mycosphere in a comparative fashion. 2.g. The bacteria inhabiting this area in soil were assumed to best represent the mycosphere microbiota. however this has been suggested to be nearly impossible due to inherent problems of incomplete desorption of cells from soil particles. the same standardized extraction protocol is used. sampling of the mycosphere has not turned into a routine practice yet. Molecular analyses of the soil and mycosphere microbiota e soil nucleic acids as the basis 3. Following the isolation and purification of soil DNA/RNA. Thus. This window is limited and biased per definition. Then. and the reader is referred to some of the relevant pioneering studies [2. the key initial desorption and lysis steps are inevitably prone to biases. In the specific case of mushroom-forming ecotomycorrhizal fungi. focussing on messenger RNA (for instance. Such approaches should go handin-hand with the aforementioned cultivation-based approaches in a polyphasic approach. 3. Boersma / European Journal of Soil Biology 47 (2011) 77e87 bacteria.G. The mycosphere serves as a habitat for diverse bacteria that.7.78 J. is actually quite difficult. Another key issue is that most (but not all) analyses have commonly targeted soil DNA instead of RNA. The interface is influenced by compounds that become available from the fungal mycelium via direct secretion of organic acids like oxalic acid or trehalose [15] or compounds like glycerol [17] or via dying fungal cells. the mycosphere poses a numbers of different problems to sampling. A sensible approach developed in our laboratory [16] sampled the mushroom foot part. We will first provide an outline of the peculiarities of the mycosphere as a particular microhabitat in the soil. bead beating based) and. either underneath fungal fruiting bodies or directly in the soil hyphal network.9. e. Furthermore. per scientific study. However. an array of analytical techniques is available to provide answers to the scientific question that is being posed.9]. However. It follows that. This definition does not clearly define the exact dimensions of the soil compartment to be sampled. and they are obviously indispensable in studies on the transcriptome. [19]). with emphasis on soil DNA The vast majority of current molecular analyses from soil is preceded by direct soil nucleic acid extractions [5]. The mycosphere can be defined as the interface between soil fungal mycelium and the (bulk) soil environment [12].H. which has been standardized across laboratories. In contrast. the mycosphere has been defined as the narrow zone of soil around the bundled hyphae (hereafter called the “bundle”) at the base of the mushroom [11. it is imminent that.15]. F. and hence such analyses should be encouraged. sampling them in a representative manner and including the proper amount of surrounding mycosphere soil. which may read as “the soil compartment which is under the influence of the hyphae of the fungus studied”. common sample sizes for molecular assessments. as each protocol will introduce its own biases with respect to quality and quantity of the extracted DNA [8. many current assessments of soil microbial communities are based on the isolation of microbial DNA or RNA directly from soil samples [7e9]. which are often less than 1 g. all cells in the sample are released and subsequently lysed in one go. in enhanced shearing of the DNA of those cells with the most fragile envelopes (those that first yield . a particular “window” at the true extant diversity in the soil habitat is obtained. is often readily sampled. inhabit this interface. on the one hand. after which we will examine current procedures for the extraction and processing of microbial nucleic acids from soil. the performance of a particular extraction kit needs to be tested and validated [5. Next to the mycosphere. Methods that allow access to soil nucleic acids originate from the eighties. In the light of the diversity of the soil microbiota and the impossibility to have a “magic agent” that captures and lyses all cells in a given sample. This stands in sharp contrast to sampling of the rhizosphere. following break-up of the cells.11]. Nucleic acid extraction. In this respect. it is important to recognize the difference between. as a specific microhabitat compartment of soil. specifically or stochastically. However. as it often pays off to have bacterial isolates handy next to direct molecular data.8]. RNA-based protocols are in use in ribosomal marker-based studies (in the light of the greater number of ribosomes than chromosomes per cell).1. several of them finetuned to soil DNA. Bead beating may result. a fact of soil scientific life is that for each “new” soil. thus obtaining a shallow layer of soil surrounding the mushroom foot (Fig.g. Table 1 gives an outline of the methods and their intricacies. all guarantee robustness with respect to the quantity and quality of the DNA that is obtained. there have been fast developments in these methods. in any study on the soil microbiota. the physical (e. 1).. are often small and fragile. Thus. Finally. sequence and quantify (targeted parts of) the microbiota. Soil versus the mycosphere e issues of microhabitat sampling Bulk soil. However. although heterogeneous in nature.7. on the other hand. Again. describe in situ activities which can be very low Problems due to cross-hybridizations with low-homology sequences Large amounts of information on total All-in-once analysis in high-throughput. direct information on sequences. different sensitivity levels Accounts of dominant sequence types in the community Stable isotope probing and BrdU High Microarrays Medium Direct information on incorporation of label into community members: highlights active bacteria Parallel information on diversity.G. High T-RFLP. Chemical integrity and purity of soil DNA may limit analyses Only species >0. TGGE. F. lacks representation. Sensitive Several PCR biases and artifacts. Relation between structure and function can be elucidated Currently very high throughput. allows diversity estimates Gives information on the active community.H. possibility of obtaining different fingerprints from same sample Easy census of target genes in community. van Elsas. Special emphasis is placed on both the promise and the potential caveats of these methods. DGGE discussed in text Pitfalls due to cloning bias DGGE has turned into a routine fingerprinting method. Methods are error-prone! Wrong interpretations and active members of the community High potential for comparative studies due to artifacts/errors at sequence level Allows high-throughput analyses across habitats Method of choice in many studies. including inhibition Only top-1000 of target community is accessed.D. Morphotypes hard to distinguish. RISA. SSCP. caution with interpretation of data needed J. allow detection and/or quantification Easy comparisons between samples. 79 . Boersma / European Journal of Soil Biology 47 (2011) 77e87 Method Cultivation (plating) Reproducibility: divided in three classes: high (average SD below 10%). Care to be taken with the interpretations due to biases Nice but limited overview of target gene/organism diversity Fingerprintings (DGGE.1e1% abundance are visible Cultivation-based analyses key support for molecularly-based observations Soil nucleic acid extraction Low resolution. medium (average SD 10e25%) and high (average SD > 25%). Reproducibility Interpretation of results Advantages Disadvantages Major pitfalls Remarks Medium Limited information on in situ active populations due to Great Plate Count Anomaly Allows to further analyze colonies including metabolic characteristics or whole genome sequence High Easy access to genes of extant soil microbial community PCR/qPCR High Snapshot of extant microbiota in the form of information-carrying molecules Proxies of organisms or genes amplified and/or quantified Only culturable microorganisms found (only 1% of community). at phylogenetic or functional levels High-throughput sequencing: -metagenome -metatranscriptome Medium Routine techniques of high sensitivity. LH-PCR) -phylogenetical -functional Clone libraries Medium Snapshot views of (dominant) microbial diversity and community make-up.Table 1 An overview of methods suitable for assessments of bacteria in soil and the mycosphere. Prone to incomplete and biased sampling Nucleic acids as the basis of all molecular work: biases need to be reduced Key method for molecular detection from soil Several pitfalls due to nature of separation techniques. Laborious preparation of sample Problems of opportunists blurring the data Only chipped genes are found Relies on activity of Widely appreciated method to microorganisms. . USA) resin-based DNA purification steps. An overview of methods that are recommended to study the diversity and function of bacterial inhabitants of the mycosphere. Furthermore. On the other hand.80 J. by nature. commercial extraction kits. similar types of potential biases will be encountered. which should be minimized [24]. the nucleic acids that are liberated may bind differently to soil particles (clay and organic matter) in soils of different texture or mineral composition. On the other hand. In both cases. as comparisons based on data from DNA extracts that underwent different purification protocols may be scientifically unwarranted in the light of possible biases between these. The required purification steps may incur losses of material.g. up to the so-called crude lysate) is commonly followed by one or more purification steps. a critical and comparative use of soil-extracted nucleic acids does provide the investigator with a very powerful data source. have been found to reproducibly yield PCRamplifiable DNA from a variety of soils [8]. Soil nucleic acid extraction (often consisting of processing a soil sample. van Elsas. and these will thus escape detection. soil DNA samples can be . in a snapshot approach. in particular Powersoil. free DNA). Thus. The type of desorption and lysis thus determines our ultimate view of the microbial community in the sample [8. it should be recognized that. incomplete and biased. which hamper subsequent analytical methods that require PCR or labelling for hybridization. as the aforementioned biases with respect to incompleteness of sampling of the extant nucleic acid diversity have not been (completely) solved. as the crude extract often still contains a substantial amount of compounds like humic or fulvic acids. in several steps. DNA/RNA extraction methods will most likely work differently. USA). those produced under the names “Ultraclean” or “Powersoil” soil DNA extraction kits (MoBio. the microbial communities that abound in the soil system of study.G.2. a note of caution should be given here. the nucleic acid extraction protocols of choice are currently based on just a few commercial kits. The combination of cultivation-independent with cultivation-dependent methods is predicted to enable great advances in our understanding of the system. 1. across soils. the mycosphere. Combined with so-called Wizard (Promega. in different soils. which was suitable for subsequent PCR amplification analysis. substantial biases are introduced in the analyses.g. This implies that investigators applying direct soil nucleic acid extraction methods accept the view that their depiction of the soil microbial community is. For instance. Ideally. F.. which supported the contention that. clayey as well as organic-matter-rich soils. Representative DNA of adequate purity and reasonably high molecular weight has consistently been obtained. It seems mandatory that attempts are made to optimize cell lysis in accordance with the soil type and the bacterial taxon that is targeted. a reassuring degree of commonality was found between bacterial communities in soils of similar texture [23]. This has included sandy. the purpose being to obtain an overall view of the soil microbial community diversity and make-up. enzymatic lysis may not affect those bacteria that are resistant to too soft lysis.H. Comparison of the microbiota in different soil types may thus be hampered by this variable DNA extraction efficiency. 3. USA) and/or the Fast DNA Spin kit for soil (Bio101. in a grossly similar extraction background.22]. Molecular analyses of the soil microbiota e pioneering studies using hybridization Early pioneering approaches set out to directly analyze microbial community nucleic acids (mainly DNA) extracted from soil. Boersma / European Journal of Soil Biology 47 (2011) 77e87 Fig. e. purification steps are harmonized across samples. In most laboratories. e. allowing him/her to directly picture. On the positive side.D. However. . PCR amplification of functional genes has allowed a depiction of the diversities of such genes. the resolving power of the 16S rRNA gene is rather low. has been taken as characteristic for the level of complexity (diversity) of the microbial communities under study. This latter problem can actually be circumvented by using group-specific primers. and hence PCR is biased against the so-called “rare biosphere”. all with their specificities in respect of reaction fidelity. Recent work of Costa et al. and this process is still ongoing [32]. which is possible once sequences are known.1.H. further processing steps that offer increased analytical power (with respect to the analysis of microbial community make-up) are required. a key enzyme in nitrogen fixation). F. Although successfully used in many soil studies (e. A recent study illustrating this.e. information on the extant sequences of the gene of choice can thus be obtained from soil nucleic acids. Liesack and Stackebrandt [31] were the first to describe a totally novel bacterial phylum. the signal is transformed into predicted target gene numbers on the basis of a pre-established calibration line with standard target DNA. alternative single-copy markers. The first criterion. .43]. Special attention to the fidelity and consistency of amplification offered by such enzymes is required. the method has a number of potential biases. the use of these genes appears as a promising approach.G. as the polymorphisms within them may well reflect evolutionary history and also contemporary diversity across the members of a targeted community [45]. proofreading activity and thermal stability.. gyrB [44] and recA [43] have been sought. PCR as the basis for highly-sensitive analytical approaches to the soil microbiota A major step forward in the study of the soil microbiota via DNA (and/or RNA) has been the development of direct PCR amplification of target genes [28e30]. Second. been primordial in the discovery of a large number of novel bacterial radiations. PCR on the basis of so-called universal bacterial primers that target (part of) a common gene like the rRNA gene will not amplify all extant bacterial diversity simply because primers used may miss a considerable part of the community [36]. it has been shown that. the a.3. Although many studies use prior soil nucleic acid based PCR. [46].g. only targets that are dominant in the sample will be amplified. methylobacteria and sphingomonads have been concocted and successfully applied [37e41]. gproteobacteria or bacilli) have been developed. 3. For this purpose. PCR underlies many nucleic acid based analytical approaches 3. That is. Quantitative PCR (qPCR) PCR of soil DNA can be used in a quantitative manner using a socalled Taqman or “real-time” approach [47]. PCR based on soil nucleic acids has turned into a basic step in soil molecular analyses. PCR can be based on soil DNA or RNA. Third. which was based on the use of the global regulator gacA as the marker to separate the pseudomonads. as indicated. In a similar fashion. that selected bacterial (sphingomonad) 81 communities were quite different in these two contrasting environments [35].2. b. with two primers that anneal at the opposite ends of the template. When it passes a certain threshold level.. the DNA polymerase used in PCR has to be resistant to high temperature. The second criterion (hybridization) has enabled investigators to assess the levels of commonality between soil ecosystems. the same PCR may yield hybrid molecules called chimeras.J. although microcultivation techniques have been partially successful [33. revealed that this marker gene gave a significantly higher resolution than the 16S rRNA gene. like the sequencing of inserts in so-called clone libraries and/or molecular fingerprinting techniques such as DGGE or T-RFLP. A range of thermally-stable DNA polymerases are currently in use. the latter following prior reverse transcription (yielding cDNA). as they reduce the complexity of the target community. However. a major problem often encountered with hybridization analyses performed directly on environmental DNA extracts is their general lack of sensitivity. So far. which means that particular targets amplify at higher rate than other ones. Some of the novel phyla. pseudomonads harboring very similar 16S rRNA genes may have quite different ecological roles and hence differ strongly in particular accessory genes elsewhere on the genome. These group-specific approaches have often provided greater insights in the ecology (dynamics) of the target groups. qPCR is plagued by the very same biases as PCR based on soil DNA extracts. researchers will provide increasingly more sequence data to the databases. the rate of reannealing of molten soil DNA. Using PCR followed by cloning/sequencing or fingerprinting approaches. the web-based programme CHECK_CHIMERA e from the amplicons prior to further analyses. Hence. Such chimeras need to be removed e using for instance. which limits these analyses to populations of cells or genes that occur in relatively high numbers in the environmental samples [27]. In a seminal paper in which PCR amplification of 16S rRNA genes was used following by cloning and sequencing. in particular for the pseudomonads. or functional gene markers like amoA (encoding ammonia monooxygenase. which allow amplification of DNA from low-abundance organisms. being PCR performed on environmental nucleic acids the obvious choice. TM7. which result from so-called “jumping PCR”. Boersma / European Journal of Soil Biology 47 (2011) 77e87 characterized in terms of their reannealing [18] and/or hybridization behavior [25]. This results in the generation of numerous copies of the region spanned by the two primers. 3. This limitation for the alternative markers hampers sequence analysis and primer design. primer sets e often consisting of nested or semi-nested systems e that target bacteria at the group level (e.g. the premise being that the more complex the microbial community is. a key enzyme in the oxidation of ammonia) or nifH (encoding nitrogenase reductase. [30]).34]. Given the fact that the 16S rRNA genes may occur in multiple copies per genome. as PCR error levels differ. on the basis of 16S rRNA gene-based PCR applied to DNA from the mycosphere versus corresponding bulk soil. primer sets targeting specific taxa like the pseudomonads. Such target genes include a phylogenetically-tuned marker such as the 16S ribosomal RNA (rRNA) gene or the rpoB gene. enhancing sequence resolution. Sensitive instruments have been developed that allow the real-time detection of the signal produced. However. in many later studies. following PCR. The principle of this method lies in the generation of a fluorescent and detectable signal by exonuclease activity of the polymerase.3.g. the limited amount of sequence information of these genes in the database stands in sharp contrast to the enormous number of 16S rRNA gene sequences that are present. are without cultured representatives to date. Signal is produced at each cycle of the PCR reaction. or even the prevalence of particular genes of interest in the community [26]. Finally. i. qPCR is currently widely applied to soil-extracted DNA. van Elsas. e. from soil. like rpoS [42. Naturally. which need strong consideration.. The method is based on the cyclic enzymatic extension of a particular gene region (using temperature-driven cycles of denaturing and annealing).3. denoted the Planctomycetales. Alternatively. Ribosomal RNA gene-based PCR analyses of soil and other environmental DNAs have.D. allowing the quantification of numbers of target genes such as 16S rRNA genes (to quantify soil bacteria) or of functional genes like amoA or nifH. the lower the rate of reannealing of completely molten soil DNA will be [18]. Given the high denaturing temperature (often 94 C). First. the perceived diversity is prone to so-called differential amplification. Conversely. revealed. g.4. as indicated before. cloning into a library followed by sequencing of selected library clones can be used. The ability to excise. To assist or finetune the direct nucleic acid based molecular fingerprinting methods.g. then multiple bands arise on gel which are provenient from the same organism. Alternatively. at the level of a functional gene (such as amoA). Given the fact that DGGE is the most frequently used technique in many soil research laboratories. Due to these issues as well as the still qualitative nature of the PCR which is used as the basis for the generation of molecules of different sequence.48]. as key rhizosphere inhabitants.48]. Direct hybridization of the amplicons to a specific probe using a dot blot or Southern blot approach is another option. 13C in substrate that can be consumed) prior to soil sampling (e. this method will be described in more depth in the following section.4. PCR-DGGE In PCR-based DGGE. [62] did not find any difference in the eubacterial communities between GM and non-GM tobacco. which yields relatively simple community fingerprints that can be used for comparative purposes. on the basis of the method. analysis of the amplified target sequences is needed to yield the data on the microbial communities that are sought. However. qPCR may be well employed at the microhabitat/mycosphere level to assess to what extent local conditions affect gene and gene expression levels. pinpointing Variovorax spp. or. were based on the use of bacterial 16S rRNA genes..4.4. In the light of the PCR biases as discussed above.G. the first DGGE and TGGE analyses that were ever performed. incorporation of label (e. is that all the soil DNA-based applications will detect both viable and non-viable (or even dead) populations of cells.. Furthermore. although this phenomenon is often detectable on gel [59]. quantification of the bands as a tool to predict the absolute abundance of particular bacterial types in the community is often of questionable value. If this microheterogeneity yields differences in melting.. alternatively. Molecular fingerprinting techniques Following PCR of either the 16S rRNA gene or any phylogenetic or functional gene of choice. Secondly. terminal restriction fragment length polymorphism (T-RFLP) [49]. .21]. [61] showed. A range of molecular fingerprinting methods based on PCRgenerated amplicons. the method will not access organisms of the rare biosphere of soil. Boersma / European Journal of Soil Biology 47 (2011) 77e87 it provides an inherently biased picture of target gene abundance and obviously does not detect any gene of the same function with aberrant sequence. 3. the formation of heteroduplexes may cause an overestimation of the number of bands present. and a matter of caution. see below) provides an emerging very promising complementary approach. However. 3.2. Thus. PCR-DGGE has been optimized for use with soil DNA in the last decade and now constitutes a routine and reliable method to produce rapid depictions of (dominant) soil microbial communities. In contrast. different sequences may display similar migratory behavior in the gel. temperature gradient gel electrophoresis (TGGE) [40. although the sizes of the underlying amplicons may limit the information that is obtained [14. e. next to Acetobacter spp.3. van Elsas. on the basis of this method.g.. as typically only up to 100 bands can be distinguished in a gel lane. F.. ephemeral minor changes in the bacterial diversity between GM and non-GM canola. All of these methods enable the direct fingerprinting of soil microbial communities at different levels of resolution. the presence of multiple melting domains within the same molecule may cause bands to appear fuzzy on gel [58].H. reamplify and sequence particular bands in the patterns even allows for the identification of the microbial types or genes that underly these bands. For one. single-strand conformational polymorphism (SSCP) [50]. Only the most abundant members of a microbial community are thus detectable. Commonly used molecular analytical approaches 3.g. [60] could clearly dissect the bacterial communities in the rhizosphere of Chrysanthemum. The techniques were originally developed for mutation detection [56]. between which microheterogeneity may exist. yielding information about the presence of regions of homology to the probe. the suitability with respect to the quick comparison of large numbers of samples from different treatments has made the technique common property across a range of laboratories. and specifically important for the 16S rRNA based approaches. ribosomal internal spacer analysis (RISA) [51] and length heterogeneity-PCR (LH-PCR) [52] have emerged. of those organisms that are actively involved in a particular ecosystem task. and among them. The advantage of these methods is that they allow a direct comparative overview of the composition and diversity of the (dominant) soil microbiota targeted. Depending on the primers used. the incorporation of BrdU under certain conditions [54] or the transformation of the compound carrying the label [55]. but they have been extensively used for soil microbial community analyses since the 90-ies [5. These two groups are hardly distinguishable without performing additional assessments.1% of the total (the “top-1000”). as well as TGGE. PCR-based molecular fingerprinting techniques have superseeded most other post-PCR analytical methods that allow insight into soil microbial diversity and community make-up. Hence. bromodeoxyuridine [BrdU] or stable isotope prelabelling of cells. Thirdly. The methods allows the pinpointing. in a microbial community.82 J. without applying any kind of deliberate “pre-bias”. respectively for DGGE and TGGE. Bacterial phylogenetic DGGE The 16S rRNA gene is nowadays routinely used in PCR-DGGE as well as TGGE as the proxy for bacterial phylogenetic relatedness. Gyamfi et al. in the 90-ies. some organisms contain several (up to 15) ribosomal operons. Application of the method to diverse soil communities has yielded important scientific insights that were impossible to achieve before the onset of the approach.D. 3. the methods are clearly limited to the dominant members (the so-called top-1000) of the microbial community that is targeted. Given the successes with such analyses. restriction fragment length polymorphism analysis (PCR-RFLP). Finally. gyrA or recA). Thus. such as analyzing cell viability in a direct viable count assay [53]. it may be possible to more precisely map microbial diversity and function to soil/mycosphere space. it has inherent limitations with respect to its resolving power. Duineveld et al. In fact. we examine to what extent phylogeneticallybased bacterial PCR-DGGE allows us to make inferences about soil microbial community make-up. Angelo-Picard et al. providing information about the diversity and nature of the sequences that are targeted. DGGE of PCR amplicons has been most widely accepted.1. Over the last decade. Hereunder. Concerning the area of the assessment of the impact of genetically-modified (GM) plants. it seems likely that 16S rRNA-based PCR-DGGE will remain one method of choice in future studies on the impact of GM plants on the soil microbiota. These further analysis can proceed via e. PCR-DGGE still faces problems which may hamper the analyses. This is achieved on polyacrylamide gels with denaturing or temperature gradients. with a threshold of roughly 0. thus giving rise to coinciding bands [57]. such as denaturing gradient gel electrophoresis (DGGE) [48]. Another observation. similar-sized amplicons generated by PCR are separated on the basis of differences in their nucleotide sequences. PCR-DGGE can depict the microbial diversity and community make-up at the level of the 16S rRNA gene (phylogenetically-based fingerprinting) or any other marker gene (such as rpoB. In spite of its current routine use and wide acceptance. and nifH encoding the dinitrogenase reductases of nitrogen-fixing bacteria [45]. DNA microarrays (chips) Over the last decade.5. as outlined in the foregoing.J. i. This way. Direct pyrosequencing or microarray analysis of soil DNA bypasses this potential bias (see sections below). The authors did not find a significant effect of the transgenic plant on the nitrogen-fixing communities. To solve the . In this method. attention was given to functions in which the genes are harbored by only one or few bacterial species. One of the greatest challenges for the forthcoming years will be to understand how microbial diversity affects the functioning of the soil system and to address the issue of stability of function in the face of stress imposed on the soil. are positioned in a dense array. which increases our knowledge of soil microbial community make-up. or a functional gene) are ligated into a suitable vector plasmid. Therefore. however. Clone libraries have high resolution but e as large samples are needed to detect the less abundant (rare) bacterial types e do not allow for a quick overview of the diversity per sample or the difference between samples. On the microarray. community shifts observed via PCR-DGGE gels based on 16S rRNA genes do not a priori provide information on soil functioning. [69. as discussed before.e. reduced soil microbial diversity might not necessarily correlate with poor soil functioning. analyses of clone libraries provides direct access to information (richness. In a highcomplexity sample such as soil. On the one hand. a major advantage of 16S rRNA gene based clone libraries is the ability to directly obtain and analyze novel sequences. Although the analysis of target genes encoding enzymes involved in key or sensitive soil processes provides better insight in (potential) soil function than that of the 16S rRNA gene. often after pre (PCR-based) amplification. nitrate reductase narG. information on the phylogenetic diversity and community make-up (Phylochip) as well as on functional potential (Geochip) of the soil is obtained in high throughput.D. This may affect the interpretation of the true microbial diversity in the system. such as in denitrification.4. The sensitivity of clone library analyses. practical considerations (as reflected in the question “what degree of novelty is presented with clone library analysis even if coverage is still low?”) have led the scientific community to also accept data obtained with smaller-sized libraries.g. evenness and nature) on the targeted gene sequences present in the extant microbiota. Disturbances influencing such groups. These methods may range from the detection and quantification of messenger RNA and/or of functional genes to the assessment of the rate of functioning. i. This is mainly so because the sequences are analyzed separately. van Elsas.. fluorescently labeled and brought into contact with a microarray. including the gene encoding ammonia monooxygenase. the data contained in these can be cross-compared between different soils or treatments using advanced statistical tools such as LIBSHUFF [67] or UniFrac [68]. Another limitation of phylogeneticallybased PCR-DGGE profiling is that it does not a priori allow us to assess soil functioning. In this analysis. allows for an in-depth analysis of microorganisms or functional genes present in a soil microbial community. were addressed. Moreover. the link between soil microbial diversity and function is still far from understood. 3. amoA.70]) or of functional genes (Geochip. quality and health. As another example. After growth of single colonies that received vectors with insert. Interestingly. which. It is. On the other hand. groupspecific PCR-DGGE analyses proved to be very useful to infer potential function and functional redundancy. in order to achieve satisfactory coverage of the extant bacterial diversity in soil ecosystems. Genes in the soil DNA that are homologous to the probes present on the chip will bind e via hybridization e at the positions of their homologous counterparts. After hybridization. nitrite reductases nirK and nirS. Various target genes have been used as proxies to track changes in soil functional gene diversity.G. the current highthroughput facilities also facilitate the direct generation of sequence diversity data on the basis of soil DNA. Clone library analysis is a somewhat laborious method. Clone libraries As mentioned in the foregoing. 3. the signals on the chip are digitally analyzed. cloned amplicons can be isolated by plasmid extraction. in the last decade an increasing focus has been placed on the analysis of proteinencoding genes involved in key ecosystem processes. as is the case with fingerprinting techniques. chimeras are routinely discarded. in particular cases such as the bacterial ammonia oxidizers. those encoding methane monooxygenases pmoA and mmoX [30]. Additionally.4. Functional gene-based DGGE to assess functional diversity As the functional redundancy among the bacteria in soil is often high. sequenced and the sequences analyzed by comparison to databases.6. This lack of resolving power has partially been solved by targeting specific groups within the community. which are monoor oligophyletic. In clone library analyses. DNA fragments are ligated into a vector plasmid with possibly differential efficiencies. the nifH gene has recently been used to study the impact of GM (Bt) white spruce on soil nitrogen-fixing communities [64]. is higher than that of the aforementioned fingerprinting techniques. e. the resulting constructs are introduced into Escherichia coli by transformation. important to recognize the cloning bias that is inherent to the technique. However. supposedly have a larger influence on soil functioning than those affecting highly-redundant groups [63]. [71]). bacterial ammonia oxidizers. Subsequently. an unrealistically high number of sequences is often required (roughly over 1500 or 2000 [66]). F. distinguishing potential sequence diversity and cross-hybridization may be problematic. Given the limitations of phylogenetic assessments.. soil DNA is. However. and hence single sequences from abundant or less abundant species (given a large enough sample size) are well detectable. In this respect. the phylogenetic profiles were found to correlate well with functional diversity. the web-based information that can be found per functional gene still does not come close to the wealth of information on 16S rRNA gene sequences. either consisting of fragments of 16S rRNA genes (Phylochip. there is a need to combine the two methods. the presence of the phlD gene encoding the production of the antagonistic compound diacetyl phloroglucinol (DAPG) by pseudomonads has been successfully tracked in soil using PCR-DGGE [65].e. Hence. Boersma / European Journal of Soil Biology 47 (2011) 77e87 Therefore. However. with respect to understanding the community diversity and phylogenetic make-up. the approach does not detect microorganisms of the rare biosphere in soil and also will not easily detect subtle changes in a microbial community. the analysis of the diversity and activity of the soil microbiota has been greatly spurred by the development of DNA microarrays and their use in hybridization assays of soil DNA. PCR-generated amplicons 83 (with preselected primers that target a selected phylogenetic proxy like the 16S rRNA gene. functional genes occurring in a wide range of bacteria. Gene databases have expanded enormously over the last decade.. thus bypassing the cloning step (see section High throughput sequencing). 3. up to tens of thousands of oligonucleotide probes.H. with current ultra-high-throughput sequence facilities. Rarefaction analysis has shown that. making robust and specific primer design for the detection of a range of functional genes feasible (see further). Such considerations are important as they provide ways to infer whether additional methods that address functioning are necessary. On the other hand. pyrosequencing and Solexa-based sequencing Just a few years ago. only the breadth of functions/genes that are already known can be assessed [72]. Its multiparallellity allows the production of hundreds of thousands to millions of 450-bp reads in just a single run. mostly bulk soils have been analyzed. the all-at-once glance at (potential) soil functioning offered by DNA microarrays is very attractive. However. template preparation and the actual capillary sequencing [73].76.D. however. as they (1) directly approach gene expression at the RNA level and (2) are independent of prior assumptions about the types of genes present. given current analytical power. hybridization quality and data evaluation are the crucial steps for an appropriate use of DNA microarrays to study the soil microbiota.79]. High throughput sequencing. is of direct use for the generation of.66. A major advantage of pyrosequencing is that. The reads that are produced are often relatively small (maximally 450 bp). allowing to distinguish perfect match (PM) from mismatch (MM) [70].76]. totally unknown organisms or genes will not be detected by using chips that are based on database sequences. [69] detected a higher degree of diversity by this approach than that observed by 16S rRNA gene based PCR followed by cloning and sequencing.66. starting with the most abundant species going down into the rare biosphere [77. van Elsas. Furthermore. partial 16S rRNA based reads. but this situation is rapidly changing. There are. a minimum of eleven or more short oligonucleotides have been designed. F.77] and/ or RNA [19]. The method is more error-prone than previous (Sanger-based) sequencing and thus special error (“noise”) detection programs are required [78]. Cross-hybridization becomes a key issue in particular when highly abundant 16S rRNA gene fragments share sequence similarity to nontarget probes resulting in weak false positive signals. a range of novel sequences was obtained on the basis of mRNA (metatranscriptome analysis). the NG sequencing techniques discussed so far have already been extensively used in the analyses of soil microbial diversity and community structure as well as gene expression (metatranscriptomics) across diverse soils [19. For instance. soon after its emergence.. Lastly. The phyloarrays that are presently available can thus complement the 16S rRNA gene based cloning and sequencing as well as community fingerprinting by PCR-DGGE or T-RFLP. It is foreseeable that.76. the soil DNA extraction method strongly determines the representation and eventual bias of the data.. In addition. In these cases. Hence.. namely the lack of representation for the extant community. namely library preparation. Boersma / European Journal of Soil Biology 47 (2011) 77e87 issue for the phylochip. They have been employed for soil studies [71] in which initial hybridizations showed to be rather insensitive.000 probes covering more than 10.G.g. Researchers have acknowledged this problem and have set out to prepare chips based directly on soil DNA. Pyrosequencing. however at lower read lengths (currently 35 bp on average). with improved sensitivity [81]. soil metatranscriptome-based analyses will actually superseed the microarray-based analyses. pers. The phyloarray approach was suitable to identify OTUs which contributed to the differences in the soils under different land use. yielding longer reads. to increase sensitivity. Thus. although mRNA was never dominant in the extracts [19]. given its ultra-high throughput and lack of biases. The data obtained can be placed in the context of (local) soil/mycosphere conditions to obtain gene level e habitat correlations.84 J. Thus.e. which directly uses soil DNA. may serve the purpose of “gap-filling” in 454-generated sequence data. geochips may contain over 24. Hence. In addition. In spite of such remaining challenges. we are currently witnessing a rapid shift from the already well-accepted molecular fingerprinting and microarraybased techniques to methods based on direct pyrosequencing of environmental metagenomic DNA or RNA [19. This method consists of multiparellel sequencing by synthesis.and C-cycle genes in antarctic soils [72].77]. 3. Microarray hybridization has enormous potential. The perfect match (PM)mismatch (MM) pair approach substantially improves the fidelity of hybridization. Limitations may arise by the human capability to analyze the immense amount of data obtained and of databases to deal with errors (noise) and to filter out the genes of interest [78]. in particular when direct pyrosequencing was used [66. comm). several highly powerful novel sequencing techniques. the overwhelming amount of sequence data obtained will require special bioinformatics software for easy sorting (binning) and analysis. i. but it has opened another box of problems. The Solexa platform even offers an orders of magnitude higher throughput of reads. hampering the analyses [80]. at this point in time. The sensitivities of the two NG sequencing platforms are mainly determined by the efficiency and unbiased nature of the sequencing. thus yielding only partial 16S rRNA sequence reads.76. 454-based pyrosequencing was applied to soil DNA [66. those related to the undefined nature of many of the new probes on the chip. were developed [73]. e. quantitative PCR and enzyme assays were used. sulphur and phosphorus cycling [71]. thus encompassing material from the whole sampled community (Vogel. due to its extreme throughput. direct pyrosequencing of soil DNA allows to dissect a system from the top to the bottom. In practical terms.7. analyses on the basis of pyrosequencing are a bit limited by the high costs of the equipment and procedure. On the other hand.. For instance. In spite of financial and other limitations. The geochip has been successfully applied to study N. This has potentially solved one problem.000 genes distributed among more than 150 functional groups involved in nitrogen.77]. evolutionarily-distant genes with similar function from previously unknown sources may remain unconsidered as databases may fail to identify such sequences. including the possibility to generate a so-called universal microarray describing soil quality or health. Prominent among them were the so-called 454-based/pyrosequencing [74] and Illumina/Solexa’s Genome Analyzer sequencing. positive detection will depend on the probes that are present on the chip and thus on pre-existing knowledge about the underlying organisms or genes. These high-throughput technologies seemed very suitable for massive parallel sequencing of metagenomes and metatranscriptomes [75]. It will possibly make arbitrary choices insuperable. preamplification (e. denoted next-generation (NG) sequencing methods. the approach was useful to overcome the problem of dominance. the soil microbial community structures were shown to shift in relation to soil pH as the main driver. Bottlenecks in microarray work include problems of robustness and the fact that they cannot generate information on new sequence types. indicating that the functional gene complement differed significantly across sampling locations and vegetation types. i. The 454 platform. . as well as Illumina sequencing bypass three bottlenecks in classical sequencing. the overshadowing of members of the rare soil biosphere by the dominant ones. in which the pyrophosphate that is released is detected in an enzymatic cascade ending in luciferase and detection of the emitted light. thereby giving novel insight into soil microbial diversity [80]. Probe development.e. many new sequences will be discovered. carbon. it is critical here which probes will be elected to make part of the chip. DeSantis et al.H. which substantiated the microarray hybridization results [72]. Given the novelty of the functional gene array. Hence. some drawbacks.e. whereas the Solexa platform. and the relationship between particular genes and soil health is not at all clear. in terms of the approaches used.g. Moreover. by rolling circle amplification) has been included.. Furthermore. i. had a more favourable niche [83]. may be considered as fairly representative for the estimated microbial community size in this compartment. such as the mycosphere. Moreover. . as a result of which the used V. However. a shallow layer of soil surrounding the mushroom foot was obtained (Fig. These examples are not exhaustive and could be complemented with examples from other labs. consisting of direct molecular methods and cultivation-based analyses. by a back-of-the-envelope calculation. the clear selective effect that plant roots exert on the microbial communities in the surrounding soil. and shaking the loose soil from the foot. it was important to finetune and apply a sphingomonad-specific PCR amplification system coupled to clone library analysis and DGGE fingerprinting to analyze whether communities of the targeted sphingomonads were selected in the mycosphere. On the basis of nucleic acids extracted from the mycosphere and corresponding bulk soil. as well as recent advances in the development of novel methods (microarrays and high-throughput metagenomic sequencing) and their application to soil samples. using molecular methods (see later). indicating the possible existence. Also. In this case. the mycosphere apparently constituted a hospitable microhabitat for the bacterial partner.82]. The latter analysis would be especially relevant. direct analysis of Sphingomonadaceae communities in the mycosphere of two fungal types and the comparison of these with bulk soil communities was a primary aim. Warmink and van Elsas [16] recently proposed that an excellent compartment representative of the mycosphere is provided by the foot of fungal fruiting bodies (mushrooms).G. Thus. it was thought to be imminent to analyze to what extent different members of this community become selected or deselected in the mycosphere.83]. was shown to impact the local populations of Variovorax paradoxus HB44 [17. further polyphasic studies performed in the mycosphere in microcosms revealed the selective effect of glycerol released by fungal hyphae in fungus (Lyophillum sp. and the specificity of the responses given by particular soil bacteria. Concluding remarks and outlook In this review. The data also revealed hitherto undetected bacterial groups. the application of fingerprinting as well as cloning methods to the mycosphere of selected soil fungi was examined [16. such as PCR-based fingerprinting and clone library analyses. In other words. In some examples of recent work in our laboratory.D. this fungus was shown to de-acidify the acid soil used to pH values over 5. On the basis of the respective soil and mycosphere DNAs. In another mycosphere study [35]. Soil pH. paradoxus strain.g. of universal versus specific fungiphiles [82]. By severing off the mushroom foot part. paradoxus like bacteria [17]. we provide an overview of currently almost traditional molecular methods. Moreover. Acknowledgements We thank Rashid Nazir for his assistance with providing material for this review. particular bacterial types were also obtained in culture.J. These studies revealed a clear “mycosphere effect” exerted by the varied fungi on the local bacterial communities. 5. where focussed research questions are being posed. both bacterial phylogenetic PCR-DGGE and clone library analysis were then successfully performed [16. (2) detection of their activities. van Elsas. the community revealed reduced complexity. Parallels of these data might be drawn with the well-known rhizosphere effect.35. consisting of dense networks of hyphae. messenger RNA-based). Using this sampling strategy. for such studies it is strongly advocated to apply a polyphasic analytical approach to analyzing soil and related systems. Use of molecular techniques to unravel bacterialefungal interactions in soil e the mycosphere Taking into account the aforementioned intricacies of the mycosphere habitat. i. next to the presence of glycerol.0. Furthermore.35. the further processing and analytical steps required to describe the local microbiota were found to be similar to those executed with DNA from bulk soil (see section on Soil nucleic acid extraction above. in situ (e. These bundles were hypothesized to concentrate the potential effect on mycosphere-associated bacteria and thus to serve as a hot spot for bacterial activity in soil.1. From the data 85 obtained. if possible. they illustrate that e as a result of the application of molecular tools e strong progress has been achieved in our understanding of the bacterial communities at the microhabitat/mycosphere level. allowing to study their responses to fungal hyphae in the soil. A more in-depth insight into the interactive processes that take place in this microhabitat should now be achievable using nucleic acid based metagenomics and metatranscriptomics analyses.H. Fig. given the overall nature of these methods applied to mixed soil microbial communities and the analytical power offered by having microorganisms in culture. Following the successful extraction of DNA from the mycosphere. Three anonymous reviewers are acknowledged for their very helpful comments. F. 1).82]. (3) isolation of organisms and interrogating their ecophysiological behavior in order to predict their in situ behaviour. strain Karsten) -associated V. as well as several other bacterial strains.35. The ability to precisely sample and dissect samples of such soil microhabitats into the key components needed for molecular analyses was a crucial and indispensable conditio-sine-qua-non in these analyses. to access the soil microbiota.e. Hence.. is present. Molecular methods applied to soil microhabitats e the mycosphere 4.82]. We posit that the large array of currently available molecular methods will even gain in analytical power if carefully applied to the proper soil microhabitats. The reduced complexity might be favourable for the dissection of the system by the aforementioned high-throughput metagenomics. Boersma / European Journal of Soil Biology 47 (2011) 77e87 4. a selective effect of the mycospheres of several fungi on the microbial communities -associated with these was found. where bundles. which should consist of: (1) analysis of soil microorganisms in a direct fashion on the basis of their DNA or RNA. The bacteria inhabiting this area in soil were assumed to provide the best access to the mycosphere microbiota. among soil bacteria. It was hypothesized that nutrientrich spots at the mycosphere might locally have incited bacterial growth leading to locally selective processes. allowed an in-depth analysis of bacterial population dynamics in soil.or metatranscriptomics-based sequencing approaches. this polyphasic approach.82] successfully isolated 2e5 mg of DNA per g of mycosphere soil. as responses of the bacterial partners to soil fungi and vice versa in the microcosm might be directly assessed from comparative analyses of the metatranscriptomes obtained from systems with or without any one of the partners.35.. A major finding was that the apparent diversity of the bacterial community in the mycosphere generally decreased. However. Warmink and co-workers [16. 1). 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