European Journal of Soil Biology 47 (2011) 77e87

Contents 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 Grifths

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.
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: j.d.van.elsas@rug.nl (J.D. van Elsas).
1164-5563/$ e see front matter 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 efciently extracted from soil and subsequently
analyzed [2,3]. The molecularly-based methods specically enable
to produce snapshots of the molecular make-up of whole
complex soil microbial communities, as well as of specic 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 identication 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 specic culturable
bacteria in soil settings. However, they were inherently limited in
their scope due to the general unculturability of a majority of soil

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J.D. van Elsas, F.G.H. Boersma / European Journal of Soil Biology 47 (2011) 77e87

bacteria. On the other hand, the examination of organisms that are

able to give a growth response is still highly crucial in many studies,
and hence such analyses should be encouraged. In contrast, many
current assessments of soil microbial communities are based on the
isolation of microbial DNA or RNA directly from soil samples [7e9].
Following the isolation and purication of soil DNA/RNA, an array of
analytical techniques is available to provide answers to the scientic question that is being posed. Such approaches should go handin-hand with the aforementioned cultivation-based approaches in
a polyphasic approach, as it often pays off to have bacterial isolates
handy next to direct molecular data.
This review will examine the molecular methods that are
currently applied to soil and mycosphere systems. We will rst
provide an outline of the peculiarities of the mycosphere as
a particular microhabitat in the soil, after which we will examine
current procedures for the extraction and processing of microbial
nucleic acids from soil. Then, relevant analytical procedures are
examined with respect to their power to detect, ngerprint,
sequence and quantify (targeted parts of) the microbiota. In
particular, advanced methods for the analysis of the diversity and
community structures of soil-, plant- and mycosphere-associated
bacterial communities are the focus [10,11]. Finally, we address the
ins and outs of the application of molecular methods to
the mycosphere, as a specic microhabitat compartment of soil.
Table 1 gives an outline of the methods and their intricacies.
2. Soil versus the mycosphere e issues of microhabitat
sampling
Bulk soil, although heterogeneous in nature, is often readily
sampled, and accepted sampling procedures based on statistical
considerations are in place. However, common sample sizes for
molecular assessments, which are often less than 1 g, still seem to
limit the scope of the investigator to the (micro)habitat that is
actually accessed. In contrast, the mycosphere poses a numbers of
different problems to sampling. The mycosphere can be dened as
the interface between soil fungal mycelium and the (bulk) soil
environment [12]. It encompasses the soil zone around the
hyphae of a range of soil fungi, e.g., soil ectomycorrhizae, arbuscular
mycorrhizae and/or saprotrophic fungi [13]. Next to the
mycosphere, which is per denition extraradical, the mycorrhizosphere e which includes inuences from plant roots e can be
recognized [11,14,15]. In the specic case of mushroom-forming
ecotomycorrhizal fungi, the mycosphere has been dened as the
narrow zone of soil around the bundled hyphae (hereafter called
the bundle) at the base of the mushroom [11,16]. The mycosphere
serves as a habitat for diverse bacteria that, specically or
stochastically, inhabit this interface. The interface is inuenced 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 does not a priori pose major problems for DNA/RNA
extractions using common procedures. However, 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. As the hyphae, either underneath fungal fruiting bodies or
directly in the soil hyphal network, are often small and fragile,
sampling them in a representative manner and including the
proper amount of surrounding mycosphere soil, is actually quite
difcult. Thus, sampling of the mycosphere has not turned into
a routine practice yet, and it is therefore not well-dened. This
stands in sharp contrast to sampling of the rhizosphere, which has
been standardized across laboratories. The difculty is also reected in the denition of the mycosphere, which may read as the soil

compartment which is under the inuence of the hyphae of the

fungus studied. This denition does not clearly dene the exact
dimensions of the soil compartment to be sampled. Thus, to
sensibly address the microbial communities of the mycosphere in
a comparative fashion, it is extremely important to consider and
apply standardized sampling procedures of the mycosphere.
A sensible approach developed in our laboratory [16] sampled the
mushroom foot part, shaking the loose soil from the foot, thus
obtaining a shallow layer of soil surrounding the mushroom foot
(Fig. 1). The bacteria inhabiting this area in soil were assumed to
best represent the mycosphere microbiota. Thus, molecular analyses applied specically to such samples in comparison to those of
the bulk soil will shed light on the specic molecular features
(phylogenetically and/or functionally) of mycosphere inhabitants.
3. Molecular analyses of the soil and mycosphere
microbiota e soil nucleic acids as the basis
3.1. Nucleic acid extraction, with emphasis on soil DNA
The vast majority of current molecular analyses from soil is
preceded by direct soil nucleic acid extractions [5]. Methods that
allow access to soil nucleic acids originate from the eighties, and
the reader is referred to some of the relevant pioneering studies
[2,7,9,18]. Such methods commonly yielded both DNA and RNA
released from soil microbial communities. Since their inception,
there have been fast developments in these methods, culminating
in the fact that current nucleic acid extraction protocols are almost
all (commercial) kit-based [8]. However, for specic purposes,
several non-kit based protocols are still in use [8]. Another key issue
is that most (but not all) analyses have commonly targeted soil DNA
instead of RNA, the reason being the greater stability of DNA upon
extraction. However, RNA-based protocols are in use in ribosomal
marker-based studies (in the light of the greater number of ribosomes than chromosomes per cell), and they are obviously indispensable in studies on the transcriptome, focussing on messenger
RNA (for instance, [19]). The commercial extraction kits for DNA,
several of them netuned to soil DNA, all guarantee robustness
with respect to the quantity and quality of the DNA that is obtained.
However, a fact of soil scientic life is that for each new soil, the
performance of a particular extraction kit needs to be tested and
validated [5,8]. Furthermore, it is imminent that, per scientic
study, the same standardized extraction protocol is used, as each
protocol will introduce its own biases with respect to quality and
quantity of the extracted DNA [8,20,21].
Key issues in soil nucleic acid extraction are the efciencies of
the release of microbial cells from soil particles and the subsequent
lysis of the former. Ideally, all cells in the sample are released and
subsequently lysed in one go, however this has been suggested to
be nearly impossible due to inherent problems of incomplete
desorption of cells from soil particles, their ready readsorption and,
nally, incomplete and biased cell lyses. 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, the key initial
desorption and lysis steps are inevitably prone to biases. It follows
that, in any study on the soil microbiota, a particular window at
the true extant diversity in the soil habitat is obtained. This window
is limited and biased per denition. Therefore, rigorous standardization is required in comparative work in soil, to keep the putative
biases similar across samples or treatments. In this respect, it is
important to recognize the difference between, on the one hand,
the physical (e.g., bead beating based) and, on the other hand, the
enzymatic (soft) cell lysis methods [2,7,9]. Bead beating may result,
following break-up of the cells, in enhanced shearing of the DNA of
those cells with the most fragile envelopes (those that rst yield

Table 1
An overview of methods suitable for assessments of bacteria in soil and the mycosphere. Special emphasis is placed on both the promise and the potential caveats of these methods.
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
amplied and/or quantied

Only culturable
microorganisms
found (only 1%
of community).
Chemical integrity and
purity of soil DNA may
limit analyses
Only species >0.1e1%
abundance are visible

Cultivation-based analyses
key support for
molecularly-based observations

Soil nucleic acid extraction

Low resolution, lacks

representation.
Morphotypes hard
to distinguish.
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. DGGE
discussed in text
Pitfalls due to
cloning bias

DGGE has turned into a routine

ngerprinting method. Care to
be taken with the interpretations
due to biases
Nice but limited overview of
target gene/organism diversity

Fingerprintings (DGGE, TGGE, High

T-RFLP, SSCP, RISA, LH-PCR)
-phylogenetical
-functional
Clone libraries
Medium

Snapshot views of (dominant)

microbial diversity and
community make-up,
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,
at phylogenetic or functional levels

High-throughput sequencing:
-metagenome
-metatranscriptome

Medium

Routine techniques of high sensitivity;

allow detection and/or quantication
Easy comparisons between samples,
possibility of obtaining different
ngerprints from same sample
Easy census of target genes
in community; allows diversity
estimates
Gives information on the active
community. Relation between
structure and function
can be elucidated
Currently very high throughput, direct
information on sequences. Sensitive

Several PCR biases and

artifacts, including
inhibition
Only top-1000 of target
community is accessed.

Laborious preparation
of sample
Problems of opportunists
blurring the data

Only chipped genes

are found

Relies on activity of
Widely appreciated method to
microorganisms,
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. 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. Again, caution with
interpretation of data needed

J.D. van Elsas, F.G.H. Boersma / European Journal of Soil Biology 47 (2011) 77e87

Method
Cultivation
(plating)

Reproducibility: divided in three classes: high (average SD below 10%), medium (average SD 10e25%) and high (average SD > 25%).

79

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J.D. van Elsas, F.G.H. Boersma / European Journal of Soil Biology 47 (2011) 77e87

Fig. 1. An overview of methods that are recommended to study the diversity and function of bacterial inhabitants of the mycosphere. The combination of cultivation-independent
with cultivation-dependent methods is predicted to enable great advances in our understanding of the system.

free DNA). On the other hand, enzymatic lysis may not affect those
bacteria that are resistant to too soft lysis, and these will thus
escape detection. In both cases, substantial biases are introduced in
the analyses. The type of desorption and lysis thus determines our
ultimate view of the microbial community in the sample [8,22]. It
seems mandatory that attempts are made to optimize cell lysis in
accordance with the soil type and the bacterial taxon that is targeted. Furthermore, it should be recognized that, in different soils,
DNA/RNA extraction methods will most likely work differently. For
instance, the nucleic acids that are liberated may bind differently to
soil particles (clay and organic matter) in soils of different texture
or mineral composition. Comparison of the microbiota in different
soil types may thus be hampered by this variable DNA extraction
efciency. On the positive side, a reassuring degree of commonality
was found between bacterial communities in soils of similar
texture [23], which supported the contention that, across soils, in
a grossly similar extraction background, similar types of potential
biases will be encountered.
Soil nucleic acid extraction (often consisting of processing a soil
sample, in several steps, up to the so-called crude lysate) is
commonly followed by one or more purication steps, as the crude
extract often still contains a substantial amount of compounds like
humic or fulvic acids, which hamper subsequent analytical
methods that require PCR or labelling for hybridization. The
required purication steps may incur losses of material, which
should be minimized [24]. Ideally, purication steps are harmonized across samples, as comparisons based on data from
DNA extracts that underwent different purication protocols
may be scientically unwarranted in the light of possible biases
between these.

In most laboratories, the nucleic acid extraction protocols of

choice are currently based on just a few commercial kits, e.g., those
produced under the names Ultraclean or Powersoil soil DNA
extraction kits (MoBio, USA) and/or the Fast DNA Spin kit for soil
(Bio101, USA). Combined with so-called Wizard (Promega, USA)
resin-based DNA purication steps, commercial extraction kits, in
particular Powersoil, have been found to reproducibly yield PCRampliable DNA from a variety of soils [8]. This has included sandy,
clayey as well as organic-matter-rich soils. Representative DNA of
adequate purity and reasonably high molecular weight has
consistently been obtained, which was suitable for subsequent PCR
amplication analysis. However, a note of caution should be given
here, as the aforementioned biases with respect to incompleteness
of sampling of the extant nucleic acid diversity have not been
(completely) solved. This implies that investigators applying direct
soil nucleic acid extraction methods accept the view that their
depiction of the soil microbial community is, by nature, incomplete
and biased. On the other hand, a critical and comparative use of
soil-extracted nucleic acids does provide the investigator with
a very powerful data source, allowing him/her to directly picture, in
a snapshot approach, the microbial communities that abound in the
soil system of study, e.g., the mycosphere.
3.2. 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, the
purpose being to obtain an overall view of the soil microbial
community diversity and make-up. Thus, soil DNA samples can be

J.D. van Elsas, F.G.H. Boersma / European Journal of Soil Biology 47 (2011) 77e87

characterized in terms of their reannealing [18] and/or hybridization behavior [25]. The rst criterion, i.e., the rate of reannealing of
molten soil DNA, has been taken as characteristic for the level of
complexity (diversity) of the microbial communities under study,
the premise being that the more complex the microbial community
is, the lower the rate of reannealing of completely molten soil DNA
will be [18]. The second criterion (hybridization) has enabled
investigators to assess the levels of commonality between soil
ecosystems, or even the prevalence of particular genes of interest in
the community [26]. However, a major problem often encountered
with hybridization analyses performed directly on environmental
DNA extracts is their general lack of sensitivity, which limits these
analyses to populations of cells or genes that occur in relatively
high numbers in the environmental samples [27]. Hence, further
processing steps that offer increased analytical power (with respect
to the analysis of microbial community make-up) are required,
being PCR performed on environmental nucleic acids the obvious
choice.
3.3. PCR underlies many nucleic acid based analytical
approaches
3.3.1. 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 amplication
of target genes [28e30]. Such target genes include a phylogenetically-tuned marker such as the 16S ribosomal RNA (rRNA) gene or
the rpoB gene, or functional gene markers like amoA (encoding
ammonia monooxygenase, a key enzyme in the oxidation of
ammonia) or nifH (encoding nitrogenase reductase, a key enzyme
in nitrogen xation). Using PCR followed by cloning/sequencing or
ngerprinting approaches, information on the extant sequences of
the gene of choice can thus be obtained from soil nucleic acids. In
a seminal paper in which PCR amplication of 16S rRNA genes was
used following by cloning and sequencing, Liesack and Stackebrandt [31] were the rst to describe a totally novel bacterial
phylum, denoted the Planctomycetales, from soil. Ribosomal RNA
gene-based PCR analyses of soil and other environmental DNAs
have, in many later studies, been primordial in the discovery of
a large number of novel bacterial radiations, and this process is still
ongoing [32]. Some of the novel phyla, e.g., TM7, are without
cultured representatives to date, although microcultivation techniques have been partially successful [33,34]. In a similar fashion,
PCR amplication of functional genes has allowed a depiction of the
diversities of such genes.
PCR can be based on soil DNA or RNA, the latter following prior
reverse transcription (yielding cDNA). The method is based on the
cyclic enzymatic extension of a particular gene region (using
temperature-driven cycles of denaturing and annealing), with two
primers that anneal at the opposite ends of the template. This
results in the generation of numerous copies of the region spanned
by the two primers. Given the high denaturing temperature (often
94
C), the DNA polymerase used in PCR has to be resistant to high
temperature. A range of thermally-stable DNA polymerases are
currently in use, all with their specicities in respect of reaction
delity, proofreading activity and thermal stability. Special attention to the delity and consistency of amplication offered by such
enzymes is required, as PCR error levels differ. PCR based on soil
nucleic acids has turned into a basic step in soil molecular analyses,
like the sequencing of inserts in so-called clone libraries and/or
molecular ngerprinting techniques such as DGGE or T-RFLP.
A recent study illustrating this, revealed, on the basis of 16S rRNA
gene-based PCR applied to DNA from the mycosphere versus corresponding bulk soil, that selected bacterial (sphingomonad)

81

communities were quite different in these two contrasting environments [35]. Although many studies use prior soil nucleic acid
based PCR, the method has a number of potential biases, which need
strong consideration. First, 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].
Second, following PCR, the perceived diversity is prone to so-called
differential amplication, which means that particular targets
amplify at higher rate than other ones. Third, the same PCR may
yield hybrid molecules called chimeras, which result from so-called
jumping PCR. Such chimeras need to be removed e using for
instance, the web-based programme CHECK_CHIMERA e from the
amplicons prior to further analyses, which is possible once
sequences are known. Finally, as indicated, only targets that are
dominant in the sample will be amplied, and hence PCR is biased
against the so-called rare biosphere. This latter problem can
actually be circumvented by using group-specic primers, which
allow amplication of DNA from low-abundance organisms. For this
purpose, primer sets e often consisting of nested or semi-nested
systems e that target bacteria at the group level (e.g., the a, b, gproteobacteria or bacilli) have been developed. Alternatively,
primer sets targeting specic taxa like the pseudomonads, methylobacteria and sphingomonads have been concocted and successfully applied [37e41]. These group-specic approaches have often
provided greater insights in the ecology (dynamics) of the target
groups, as they reduce the complexity of the target community.
Given the fact that the 16S rRNA genes may occur in multiple
copies per genome, alternative single-copy markers, like rpoS
[42,43], gyrB [44] and recA [43] have been sought. So far, the use of
these genes appears as a promising approach, as the polymorphisms within them may well reect evolutionary history and
also contemporary diversity across the members of a targeted
community [45]. However, 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. This limitation for the alternative markers
hampers sequence analysis and primer design. Naturally,
researchers will provide increasingly more sequence data to the
databases, enhancing sequence resolution. Conversely, it has been
shown that, in particular for the pseudomonads, the resolving
power of the 16S rRNA gene is rather low. That is, 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. Recent work of Costa et al. [46],
which was based on the use of the global regulator gacA as the
marker to separate the pseudomonads, revealed that this marker
gene gave a signicantly higher resolution than the 16S
rRNA gene.
3.3.2. Quantitative PCR (qPCR)
PCR of soil DNA can be used in a quantitative manner using a socalled Taqman or real-time approach [47]. The principle of this
method lies in the generation of a uorescent and detectable signal
by exonuclease activity of the polymerase. Signal is produced at
each cycle of the PCR reaction. Sensitive instruments have been
developed that allow the real-time detection of the signal
produced. When it passes a certain threshold level, the signal is
transformed into predicted target gene numbers on the basis of
a pre-established calibration line with standard target DNA. qPCR is
currently widely applied to soil-extracted DNA, allowing the
quantication of numbers of target genes such as 16S rRNA genes
(to quantify soil bacteria) or of functional genes like amoA or nifH.
Although successfully used in many soil studies (e.g., [30]), qPCR is
plagued by the very same biases as PCR based on soil DNA extracts;

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it provides an inherently biased picture of target gene abundance

and obviously does not detect any gene of the same function with
aberrant sequence. However, qPCR may be well employed at the
microhabitat/mycosphere level to assess to what extent local
conditions affect gene and gene expression levels. Thus, it may be
possible to more precisely map microbial diversity and function to
soil/mycosphere space.
3.4. Commonly used molecular analytical approaches
3.4.1. Molecular ngerprinting techniques
Following PCR of either the 16S rRNA gene or any phylogenetic
or functional gene of choice, analysis of the amplied target
sequences is needed to yield the data on the microbial communities
that are sought. These further analysis can proceed via e.g.,
restriction fragment length polymorphism analysis (PCR-RFLP),
which yields relatively simple community ngerprints that can be
used for comparative purposes. Direct hybridization of the amplicons to a specic probe using a dot blot or Southern blot approach is
another option, yielding information about the presence of regions
of homology to the probe. Alternatively, cloning into a library followed by sequencing of selected library clones can be used,
providing information about the diversity and nature of the
sequences that are targeted. Over the last decade, PCR-based
molecular ngerprinting techniques have superseeded most other
post-PCR analytical methods that allow insight into soil microbial
diversity and community make-up. The advantage of these
methods is that they allow a direct comparative overview of the
composition and diversity of the (dominant) soil microbiota targeted. A range of molecular ngerprinting methods based on PCRgenerated amplicons, such as denaturing gradient gel electrophoresis (DGGE) [48], temperature gradient gel electrophoresis (TGGE)
[40,48], terminal restriction fragment length polymorphism
(T-RFLP) [49], single-strand conformational polymorphism (SSCP)
[50], ribosomal internal spacer analysis (RISA) [51] and length
heterogeneity-PCR (LH-PCR) [52] have emerged. All of these
methods enable the direct ngerprinting of soil microbial
communities at different levels of resolution, and among them,
DGGE of PCR amplicons has been most widely accepted. In the light
of the PCR biases as discussed above, the methods are clearly
limited to the dominant members (the so-called top-1000) of the
microbial community that is targeted. Hence, without applying any
kind of deliberate pre-bias, the method will not access organisms
of the rare biosphere of soil. Another observation, and a matter of
caution, is that all the soil DNA-based applications will detect both
viable and non-viable (or even dead) populations of cells. These
two groups are hardly distinguishable without performing additional assessments, such as analyzing cell viability in a direct viable
count assay [53].
To assist or netune the direct nucleic acid based molecular
ngerprinting methods, incorporation of label (e.g., 13C in substrate
that can be consumed) prior to soil sampling (e.g., bromodeoxyuridine [BrdU] or stable isotope prelabelling of cells, see below)
provides an emerging very promising complementary approach.
The methods allows the pinpointing, in a microbial community, of
those organisms that are actively involved in a particular ecosystem
task, e.g. the incorporation of BrdU under certain conditions [54] or
the transformation of the compound carrying the label [55]. Given
the fact that DGGE is the most frequently used technique in many
soil research laboratories, this method will be described in more
depth in the following section.
3.4.2. PCR-DGGE
In PCR-based DGGE, as well as TGGE, similar-sized amplicons
generated by PCR are separated on the basis of differences in their

nucleotide sequences. This is achieved on polyacrylamide gels with

denaturing or temperature gradients, respectively for DGGE and
TGGE. The techniques were originally developed for mutation
detection [56], but they have been extensively used for soil
microbial community analyses since the 90-ies [5,48]. 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. Depending
on the primers used, PCR-DGGE can depict the microbial diversity
and community make-up at the level of the 16S rRNA gene
(phylogenetically-based ngerprinting) or any other marker gene
(such as rpoB, gyrA or recA), or, alternatively, at the level of a functional gene (such as amoA). Furthermore, 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. The ability to excise, reamplify and
sequence particular bands in the patterns even allows for the
identication of the microbial types or genes that underly these
bands, although the sizes of the underlying amplicons may limit the
information that is obtained [14,21]. In spite of its current routine
use and wide acceptance, PCR-DGGE still faces problems which
may hamper the analyses. For one, different sequences may display
similar migratory behavior in the gel, thus giving rise to coinciding
bands [57]. Secondly, the presence of multiple melting domains
within the same molecule may cause bands to appear fuzzy on gel
[58]. Thirdly, and specically important for the 16S rRNA based
approaches, some organisms contain several (up to 15) ribosomal
operons, between which microheterogeneity may exist. If this
microheterogeneity yields differences in melting, then multiple
bands arise on gel which are provenient from the same organism.
Finally, the formation of heteroduplexes may cause an overestimation of the number of bands present, although this
phenomenon is often detectable on gel [59]. 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, quantication of the bands as a tool to predict the absolute abundance of
particular bacterial types in the community is often of questionable
value. Hereunder, we examine to what extent phylogeneticallybased bacterial PCR-DGGE allows us to make inferences about soil
microbial community make-up.
3.4.3. 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. In
fact, the rst DGGE and TGGE analyses that were ever performed, in
the 90-ies, were based on the use of bacterial 16S rRNA genes.
Application of the method to diverse soil communities has yielded
important scientic insights that were impossible to achieve before
the onset of the approach. Thus, on the basis of this method, Duineveld et al. [60] could clearly dissect the bacterial communities in
the rhizosphere of Chrysanthemum, pinpointing Variovorax spp.,
next to Acetobacter spp., as key rhizosphere inhabitants. Concerning
the area of the assessment of the impact of genetically-modied
(GM) plants, Gyam et al. [61] showed, on the basis of the method,
ephemeral minor changes in the bacterial diversity between GM
and non-GM canola. In contrast, Angelo-Picard et al. [62] did not
nd any difference in the eubacterial communities between GM
and non-GM tobacco. Given the successes with such analyses, 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. However, as indicated before, it has inherent
limitations with respect to its resolving power, as typically only up
to 100 bands can be distinguished in a gel lane. Only the most
abundant members of a microbial community are thus detectable,
with a threshold of roughly 0.1% of the total (the top-1000).

J.D. van Elsas, F.G.H. Boersma / European Journal of Soil Biology 47 (2011) 77e87

Therefore, the approach does not detect microorganisms of the rare

biosphere in soil and also will not easily detect subtle changes in
a microbial community. This lack of resolving power has partially
been solved by targeting specic groups within the community, as
outlined in the foregoing. Another limitation of phylogeneticallybased PCR-DGGE proling is that it does not a priori allow us to
assess soil functioning. On the other hand, in particular cases such
as the bacterial ammonia oxidizers, the phylogenetic proles were
found to correlate well with functional diversity. Hence, groupspecic PCR-DGGE analyses proved to be very useful to infer
potential function and functional redundancy. Such considerations
are important as they provide ways to infer whether additional
methods that address functioning are necessary. These methods
may range from the detection and quantication of messenger RNA
and/or of functional genes to the assessment of the rate of
functioning.
3.4.4. Functional gene-based DGGE to assess functional diversity
As the functional redundancy among the bacteria in soil is often
high, community shifts observed via PCR-DGGE gels based on 16S
rRNA genes do not a priori provide information on soil functioning,
quality and health. In this respect, reduced soil microbial diversity
might not necessarily correlate with poor soil functioning. Given
the limitations of phylogenetic assessments, in the last decade an
increasing focus has been placed on the analysis of proteinencoding genes involved in key ecosystem processes. On the one
hand, attention was given to functions in which the genes are
harbored by only one or few bacterial species, i.e., which are monoor oligophyletic. Disturbances inuencing such groups, e.g., bacterial ammonia oxidizers, supposedly have a larger inuence on soil
functioning than those affecting highly-redundant groups [63]. On
the other hand, functional genes occurring in a wide range of
bacteria, such as in denitrication, were addressed. Gene databases
have expanded enormously over the last decade, making robust
and specic primer design for the detection of a range of functional
genes feasible (see further). However, 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. Various
target genes have been used as proxies to track changes in soil
functional gene diversity, including the gene encoding ammonia
monooxygenase, amoA, those encoding methane monooxygenases
pmoA and mmoX [30], nitrate reductase narG, nitrite reductases
nirK and nirS, and nifH encoding the dinitrogenase reductases of
nitrogen-xing bacteria [45]. Interestingly, the nifH gene has
recently been used to study the impact of GM (Bt) white spruce on
soil nitrogen-xing communities [64]. The authors did not nd
a signicant effect of the transgenic plant on the nitrogen-xing
communities. As another example, 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].
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, the link
between soil microbial diversity and function is still far from
understood. 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.
3.5. Clone libraries
As mentioned in the foregoing, analyses of clone libraries
provides direct access to information (richness, evenness and
nature) on the targeted gene sequences present in the extant
microbiota. In clone library analyses, PCR-generated amplicons

83

(with preselected primers that target a selected phylogenetic

proxy like the 16S rRNA gene, or a functional gene) are ligated into
a suitable vector plasmid. Subsequently, the resulting constructs
are introduced into Escherichia coli by transformation. After
growth of single colonies that received vectors with insert, cloned
amplicons can be isolated by plasmid extraction, sequenced and
the sequences analyzed by comparison to databases. In this
analysis, chimeras are routinely discarded, as discussed before. The
sensitivity of clone library analyses, with respect to understanding
the community diversity and phylogenetic make-up, is higher
than that of the aforementioned ngerprinting techniques. This is
mainly so because the sequences are analyzed separately, and
hence single sequences from abundant or less abundant species
(given a large enough sample size) are well detectable. Additionally, a major advantage of 16S rRNA gene based clone libraries is
the ability to directly obtain and analyze novel sequences, which
increases our knowledge of soil microbial community make-up.
Rarefaction analysis has shown that, in order to achieve satisfactory coverage of the extant bacterial diversity in soil ecosystems,
an unrealistically high number of sequences is often required
(roughly over 1500 or 2000 [66]). However, practical considerations (as reected in the question what degree of novelty is
presented with clone library analysis even if coverage is still
low?) have led the scientic community to also accept data
obtained with smaller-sized libraries. Moreover, 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].
Clone library analysis is a somewhat laborious method, which,
with current ultra-high-throughput sequence facilities, allows for
an in-depth analysis of microorganisms or functional genes present
in a soil microbial community. However, the current highthroughput facilities also facilitate the direct generation of
sequence diversity data on the basis of soil DNA, thus bypassing the
cloning step (see section High throughput sequencing). 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, as is the case with ngerprinting techniques.
Therefore, there is a need to combine the two methods. It is,
however, important to recognize the cloning bias that is inherent to
the technique, i.e., DNA fragments are ligated into a vector plasmid
with possibly differential efciencies. This may affect the interpretation of the true microbial diversity in the system. Direct
pyrosequencing or microarray analysis of soil DNA bypasses this
potential bias (see sections below).
3.6. DNA microarrays (chips)
Over the last decade, 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. In this method, soil DNA is, often after pre (PCR-based)
amplication, uorescently labeled and brought into contact with
a microarray. On the microarray, up to tens of thousands of
oligonucleotide probes, either consisting of fragments of 16S rRNA
genes (Phylochip; [69,70]) or of functional genes (Geochip; [71]),
are positioned in a dense array. 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, the signals on the chip are digitally analyzed.
This way, 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. In a highcomplexity sample such as soil, distinguishing potential sequence
diversity and cross-hybridization may be problematic. To solve the

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J.D. van Elsas, F.G.H. Boersma / European Journal of Soil Biology 47 (2011) 77e87

issue for the phylochip, a minimum of eleven or more short

oligonucleotides have been designed, allowing to distinguish
perfect match (PM) from mismatch (MM) [70]. 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. The perfect match (PM)mismatch (MM) pair approach substantially improves the delity
of hybridization. The phyloarray approach was suitable to identify
OTUs which contributed to the differences in the soils under
different land use. DeSantis et al. [69] detected a higher degree of
diversity by this approach than that observed by 16S rRNA gene
based PCR followed by cloning and sequencing. Moreover, the
approach was useful to overcome the problem of dominance, i.e.,
the overshadowing of members of the rare soil biosphere by the
dominant ones. The phyloarrays that are presently available can
thus complement the 16S rRNA gene based cloning and
sequencing as well as community ngerprinting by PCR-DGGE or T-RFLP. In addition, geochips may contain over 24,000 probes
covering more than 10,000 genes distributed among more than
150 functional groups involved in nitrogen, carbon, sulphur and
phosphorus cycling [71]. They have been employed for soil studies
[71] in which initial hybridizations showed to be rather insensitive. Hence, to increase sensitivity, preamplication (e.g., by rolling
circle amplication) has been included. The geochip has been
successfully applied to study N- and C-cycle genes in antarctic
soils [72], indicating that the functional gene complement differed
signicantly across sampling locations and vegetation types. Given
the novelty of the functional gene array, quantitative PCR and
enzyme assays were used, which substantiated the microarray
hybridization results [72].
Microarray hybridization has enormous potential, including the
possibility to generate a so-called universal microarray describing
soil quality or health. However, it is critical here which probes will
be elected to make part of the chip, and the relationship between
particular genes and soil health is not at all clear. Furthermore,
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. Hence, totally unknown organisms or genes
will not be detected by using chips that are based on database
sequences. Researchers have acknowledged this problem and have
set out to prepare chips based directly on soil DNA, thus encompassing material from the whole sampled community (Vogel, pers.
comm). This has potentially solved one problem, namely the lack of
representation for the extant community, but it has opened another
box of problems, i.e., those related to the undened nature of many
of the new probes on the chip.
Probe development, hybridization quality and data evaluation
are the crucial steps for an appropriate use of DNA microarrays to
study the soil microbiota. Bottlenecks in microarray work include
problems of robustness and the fact that they cannot generate
information on new sequence types. Thus, only the breadth of
functions/genes that are already known can be assessed [72]. In
spite of such remaining challenges, the all-at-once glance at
(potential) soil functioning offered by DNA microarrays is
very attractive. The data obtained can be placed in the context of
(local) soil/mycosphere conditions to obtain gene level e habitat
correlations.
3.7. High throughput sequencing- pyrosequencing
and Solexa-based sequencing
Just a few years ago, several highly powerful novel sequencing
techniques, denoted next-generation (NG) sequencing methods,
were developed [73]. Prominent among them were the so-called
454-based/pyrosequencing [74] and Illumina/Solexas Genome
Analyzer sequencing. These high-throughput technologies seemed

very suitable for massive parallel sequencing of metagenomes and

metatranscriptomes [75]. For instance, soon after its emergence,
454-based pyrosequencing was applied to soil DNA [66,76,77] and/
or RNA [19]. This method consists of multiparellel sequencing by
synthesis, in which the pyrophosphate that is released is detected
in an enzymatic cascade ending in luciferase and detection of the
emitted light. Pyrosequencing, as well as Illumina sequencing
bypass three bottlenecks in classical sequencing, namely library
preparation, template preparation and the actual capillary
sequencing [73]. Its multiparallellity allows the production of
hundreds of thousands to millions of 450-bp reads in just a single
run. The Solexa platform even offers an orders of magnitude higher
throughput of reads, however at lower read lengths (currently
35 bp on average).
The sensitivities of the two NG sequencing platforms are mainly
determined by the efciency and unbiased nature of the
sequencing, which directly uses soil DNA. Hence, the soil DNA
extraction method strongly determines the representation and
eventual bias of the data. The 454 platform, yielding longer reads, is
of direct use for the generation of, e.g., partial 16S rRNA based reads,
whereas the Solexa platform, due to its extreme throughput, may
serve the purpose of gap-lling in 454-generated sequence data.
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 lter out the genes of interest [78].
In practical terms, evolutionarily-distant genes with similar
function from previously unknown sources may remain unconsidered as databases may fail to identify such sequences. On the
other hand, given current analytical power, direct pyrosequencing
of soil DNA allows to dissect a system from the top to the bottom,
i.e., starting with the most abundant species going down into the
rare biosphere [77,79]. A major advantage of pyrosequencing is
that, given its ultra-high throughput and lack of biases, many new
sequences will be discovered, thereby giving novel insight into soil
microbial diversity [80]. There are, however, some drawbacks. The
reads that are produced are often relatively small (maximally
450 bp), thus yielding only partial 16S rRNA sequence reads. The
method is more error-prone than previous (Sanger-based)
sequencing and thus special error (noise) detection programs are
required [78]. Lastly, the overwhelming amount of sequence data
obtained will require special bioinformatics software for easy
sorting (binning) and analysis. It will possibly make arbitrary
choices insuperable, hampering the analyses [80]. Furthermore, at
this point in time, analyses on the basis of pyrosequencing are a bit
limited by the high costs of the equipment and procedure, but this
situation is rapidly changing.
In spite of nancial and other limitations, 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
[19,66,76,77]. In these cases, mostly bulk soils have been analyzed.
For instance, the soil microbial community structures were shown
to shift in relation to soil pH as the main driver, in particular when
direct pyrosequencing was used [66,76]. In addition, a range of
novel sequences was obtained on the basis of mRNA (metatranscriptome analysis), although mRNA was never dominant in the
extracts [19]. It is foreseeable that, with improved sensitivity [81],
soil metatranscriptome-based analyses will actually superseed the
microarray-based analyses, as they (1) directly approach gene
expression at the RNA level and (2) are independent of prior
assumptions about the types of genes present. Thus, in terms of the
approaches used, we are currently witnessing a rapid shift from the
already well-accepted molecular ngerprinting and microarraybased techniques to methods based on direct pyrosequencing of
environmental metagenomic DNA or RNA [19,66,76,77].

J.D. van Elsas, F.G.H. Boersma / European Journal of Soil Biology 47 (2011) 77e87

4. Molecular methods applied to soil microhabitats e the

mycosphere
4.1. Use of molecular techniques to unravel bacterialefungal
interactions in soil e the mycosphere
Taking into account the aforementioned intricacies of the
mycosphere habitat, 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), where
bundles, consisting of dense networks of hyphae, is present. 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. By severing off the mushroom foot part,
and shaking the loose soil from the foot, a shallow layer of soil
surrounding the mushroom foot was obtained (Fig. 1). The bacteria
inhabiting this area in soil were assumed to provide the best access
to the mycosphere microbiota. Using this sampling strategy, Warmink and co-workers [16,35,82] successfully isolated 2e5 mg of
DNA per g of mycosphere soil, which, by a back-of-the-envelope
calculation, may be considered as fairly representative for the
estimated microbial community size in this compartment.
Following the successful extraction of DNA from the mycosphere,
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; Fig. 1).
On the basis of nucleic acids extracted from the mycosphere and
corresponding bulk soil, both bacterial phylogenetic PCR-DGGE and
clone library analysis were then successfully performed [16,35,82].
These studies revealed a clear mycosphere effect exerted by the
varied fungi on the local bacterial communities. A major nding
was that the apparent diversity of the bacterial community in the
mycosphere generally decreased. In other words, the community
revealed reduced complexity. It was hypothesized that nutrientrich spots at the mycosphere might locally have incited bacterial
growth leading to locally selective processes. The reduced
complexity might be favourable for the dissection of the system by
the aforementioned high-throughput metagenomics- or metatranscriptomics-based sequencing approaches.
Furthermore, particular bacterial types were also obtained in
culture, allowing to study their responses to fungal hyphae in the
soil. Soil pH, next to the presence of glycerol, was shown to impact
the local populations of Variovorax paradoxus HB44 [17,83]. Thus,
this polyphasic approach, consisting of direct molecular methods
and cultivation-based analyses, allowed an in-depth analysis of
bacterial population dynamics in soil. The power of the ngerprinting and clone library based methods that were used in dissecting the bacterial communities in mycosphere microhabitats
was thus indicated [16,35,82].
5. Concluding remarks and outlook
In this review, we provide an overview of currently almost
traditional molecular methods, such as PCR-based ngerprinting
and clone library analyses, to access the soil microbiota, as well as
recent advances in the development of novel methods (microarrays
and high-throughput metagenomic sequencing) and their application to soil samples. 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, such as the
mycosphere, where focussed research questions are being posed. In
some examples of recent work in our laboratory, the application of
ngerprinting as well as cloning methods to the mycosphere of
selected soil fungi was examined [16,35,82]. From the data

85

obtained, a selective effect of the mycospheres of several fungi on

the microbial communities -associated with these was found,
indicating the possible existence, among soil bacteria, of universal
versus specic fungiphiles [82]. Parallels of these data might be
drawn with the well-known rhizosphere effect, i.e., the clear
selective effect that plant roots exert on the microbial communities
in the surrounding soil, and the specicity of the responses given by
particular soil bacteria. In another mycosphere study [35], 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. Hence, it was important to netune and apply a sphingomonad-specic PCR amplication system
coupled to clone library analysis and DGGE ngerprinting to
analyze whether communities of the targeted sphingomonads
were selected in the mycosphere. Moreover, it was thought to be
imminent to analyze to what extent different members of this
community become selected or deselected in the mycosphere. On
the basis of the respective soil and mycosphere DNAs, we thus
obtained an in-depth analysis of the respective sphingomonad
communities and revealed strong mycosphere effects on these. The
data also revealed hitherto undetected bacterial groups. These
examples are not exhaustive and could be complemented with
examples from other labs. However, 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. 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. Moreover, further polyphasic studies performed in the mycosphere in microcosms
revealed the selective effect of glycerol released by fungal hyphae in
fungus (Lyophillum sp. strain Karsten) -associated V. paradoxus like
bacteria [17]. Also, this fungus was shown to de-acidify the acid soil
used to pH values over 5.0, as a result of which the used V. paradoxus strain, as well as several other bacterial strains, had a more
favourable niche [83]. In this case, the mycosphere apparently
constituted a hospitable microhabitat for the bacterial partner.
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. The
latter analysis would be especially relevant, 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.
However, given the overall nature of these methods applied to
mixed soil microbial communities and the analytical power offered
by having microorganisms in culture, for such studies it is strongly
advocated to apply a polyphasic analytical approach to analyzing
soil and related systems, which should consist of:
(1) analysis of soil microorganisms in a direct fashion on the basis
of their DNA or RNA, using molecular methods (see later),
(2) detection of their activities, if possible, in situ (e.g., messenger
RNA-based),
(3) isolation of organisms and interrogating their ecophysiological
behavior in order to predict their in situ behaviour.

Acknowledgements
We thank Rashid Nazir for his assistance with providing material for this review. Three anonymous reviewers are acknowledged
for their very helpful comments.

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