REVIEW ARTICLE Plant Sciences Feed 2011 - 1 (2) : 45- 64
PLANT SCIENCES FEED ISSN : 2231 - 1971 Jounal Homepage : http://psf.lifescifeed.com
MOLECULAR METHODS IN RHIZOSPHERE MICROBIOLOGY. B. DEB ROY A UT HOR A F F I LIAT ION S
ABSTRACT
Microbial Ecology Laboratory, Department of Life Science, Assam University, Silchar-788011, Assam, India.
New molecular methods that have been used in the study of rhizosphere microorganisms are based on culture-independent assays and sophisticated molecular techniques. This paper discusses a number of the new methodologies
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techniques. The first part of this paper further comprises a thorough treatise of the
developed for detection and localization of specific bacterial populations using modern electron and fluorescence microscopy combined with specific tagging recent development of reporter gene technology, i.e. using specific reporter bacteria to detect microscale distributions of rhizosphere compounds such as nutrients, metals and organic exudates or contaminants. Second half of the paper is devoted to microbial community analysis containing a thorough treatise of nucleotide- and PCR based technologies to study composition and diversity of indigenous bacteria in the natural rhizosphere. The most recent developments of functional gene and gene expression analyses in the rhizosphere based on specific
KEYWORDS Metagenome, Microarray, Microscopy, Reporter, Proteome, Transcriptome
mRNA transcript or transcriptome analysis, proteome analysis and construction of metagenomic libraries are also included. © 2011 LifeSciFeed Ventures
specific
INTRODUCTION
reporter
bacteria
to detect
microscale
distributions of rhizosphere compounds such as
methodologies that have already been or will soon be
nutrients, metals and organic exudates or contaminants.
applicable in rhizosphere microbiology were mainly
Second half of the paper is devoted to microbial
discussed with a emphasis on rhizosphere bacteria and
community analysis containing a thorough treatise of
this paper first describes a number of the new
nucleotide- and PCR based technologies to study
methodologies developed for detection and localization
composition and diversity of indigenous bacteria in the
of specific bacterial populations using modern electron
natural rhizosphere. Also included are the most recent
and fluorescence microscopy combined with specific
developments of functional gene and gene expression
tagging techniques. First half of the paper further
analyses in the rhizosphere based on specific mRNA
comprises
transcript or transcriptome analysis, proteome analysis
a
thorough
treatise
of
the
recent
development of reporter gene technology, i.e. using
and construction of metagenomic libraries.
45
The culture-independent assays and molecular
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. SINGLE-CELL STUDIES IN THE RHIZOSPHERE BY
Sample preparation for conventional SEM involves dehydration, but the so-called Environmental Scanning
MODERN MICROSCOPICAL METHODS
Electron Microscopy (ESEM) operates slightly above the
Direct microscopy
saturation vapor pressure of water in the specimen
The approaches of resin-imbedding and thin-
chamber. Under such conditions, water remains a liquid
sectioning, development of root-growth chambers
and hydrated biological specimen may be observed
(rhizosphere microcosms), and gentle root-washing
without prior preparation. The technique has been used
techniques assuring a minimum of change for microbial
to study mineral composition in the rhizosphere
activity at the root surface per se (rhizoplane), have all
(Cabala and Teper, 2007). Although the technique has
been introduced as alternatives to direct observations
not yet been applied in rhizosphere microbiology, it
in the native rhizosphere environment. Microscopy can
may soon become useful for high resolution studies of
be used in direct visualization using e.g. scanning
microorganisms in undistorted rhizosphere samples.
electron microscopy (SEM) or combined with staining of the indigenous microbial community. In addition, microscopy
can
be
used
to
monitor
specific
Molecular stains Direct detection of single cells in a complex
microorganisms, e.g. growth activity and survival of
environment
as
the
bacterial reporter strains. In this section we will dicuss
development of high-resolution technologies primarily
a broad overview of these three major approaches to
based on unique molecular staining and tagging
study rhizosphere microbiology.
systems
for bacteria
rhizosphere
combined
has
required
with advanced
fluorescence microscopy. The molecular staining
Traditional and environmental SEM
techniques combined with fluorescence microscopy are
The porous matrix of the rhizosphere soil (or bulk
often specific enough to localize complete genera or
soil) may actually be accessible by SEM, provided the
functional groups of the indigenous microorganisms in
preparation includes imbedding in a resin, followed by
rhizosphere samples, as described below.
thin-sectioning Altemuller,
of
the
1990).
specimen et
Fukui
al.,
(Postma
and
(1994)
used
conventional SEM technique to study bacterial colonization of sugar beet seeds, and observed different
for SEM, conventional epi fluorescence
microscopy (EFM) in rhizosphere samples has always been hampered by the sample destruction necessary to
Pseudomonas putida strains, respectively. Similarly,
prepare a thin specimen for staining and observation.
Dandurand et al., (1997) obtained detailed information
Hence, sample mounting in resin and subsequent thin-
on
fluorescens
sectioning to provide structurally intact specimen is
biocontrol strain on young pea root surfaces. This
also important in fluorescence microscopy (Eickhorst
approach gave evidence of a spatial pattern for root
and Tippkotter, 2008). Where mechanical distortion
colonization including cell aggregate formation along
must be avoided or kept at a minimum, an alternative is
the entire root. Finally, Fakhouri et al., (2001) studied
the use of long-distance objectives; such objectives may
the ultra-structure of root colonization, including
in some cases provide adequate depth of the working
Pseudomonas
range for “thick” rhizosphere samples (Thrane et al.,
patterns
distributions
antagonistic
of
for
a
interactions
subtilis
As
and
distribution
Bacillus
General cell stains
Pseudomonas
between
fluorescens and pathogenic Fusarium oxysporum on
2000).
46 46
tomato roots.
Plant Sciences Feed Vol 1 Issue 4
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. Applications of direct epifluorescence microscopy
in the following. Modern fluorescence microscopy has
of soil and rhizosphere specimen include DNA staining
thus provided an excellent insight on spatial and
with acridine orange (AO) in thin-sectioned soil
temporal colonization patterns for a large number of
samples as reported by DeLeo et al., (1997). For direct
specific bacterial inoculants, i.e. from the early binding
EFM of a root specimen, the AO may be useful to stain
of inoculant cells to a seed and to their firm
the total microbial population. One such application
establishment within roots (endophytic bacteria), in the
documented a particularly high density of bacteria near
rhizoplane, or in the rhizosphere including the mucigel
root sections presumed to release specific exudates
polymer matrix or root-adhering soil.
(sucrose or tryptophan) at the root tip or older root segments, respectively (Jaeger et al., 1999). Since the hydrophobic AO compound has a strong adsorption affinity to soil humic material, unspecific binding to the soil matrix may actually be exploited as a counter stain. AO counterstaining for observation of fungal zoospores (Pythium aphanidermatum) on cucumber roots has also been used successfully (Zhou and Paulitz, 1993). An alternative, the UV-excitable DNA stain DAPI may also stain unspecifically in some soils. Where this is a
The first applications of CLSM to study bacterial root colonization were based on strain-specific fluorescent antibody (FA) staining to follow the inoculants (Schloter et al., 1993; Hansen et al., 1997). Co-inoculation
experiments
demonstrated
that
Azospirillum brasilense strains colonizing wheat roots were mutually competitive (Kirchhof et al., 1997), while Pseudomonas fluorescens strains colonizing barley roots were not (Hansen et al., 1997).
problem, a useful DNA stain is the green fluorescent
Fluorescence probing of rhizosphere bacteria with
SYBR Green II (Weinbauer et al., 1998). Finally, a useful
rRNA-targeting oligonucleotides may result in higher
stain is also Fluorescent Brightener (FB) 28 (sometimes
hybridization signals assuming that cellular rRNA
referred to as Calcofluor White M2R), applied for wall
contents correlate with growth activity (Assmus et al.,
staining of fungi in root-soil microcosms (Thrane et al.,
1997). Hence, the rapid development of novel
1999) and imbedded thin-sections of soil. A more recent Fluorescent Brigthener Agent (FBA) 220 may actually improve staining of the soil thin-sections since crystallization problems can be avoided (Harris et al.,
fluorescence in situ hybridization (FISH) probes, staining technologies and CLSM application has resulted in numerous studies of root colonization, as exemplified here. Taxonomic probes for a selected strain or group of
2002). Today these general strains are rarely used
organisms
can
be
applied,
sometimes
even
alone, but rather to gather additional information in
physiological probes for specific cellular activity.
combination with specific cell stains.
Important early observations were the 3-D patterns of active sub-populations of both inoculant and indigenous
Specific cell stains
bacteria on root surfaces. The early works by Assmus et al., (1995, 1997) using combinations of strain-specific
introduced the possibility of 3-D reconstructions to
monoclonal antibody (Azospirillum brasilense Wa3),
obtain high resolution information on the structural and
species specific FISH probe (A. brasilense), group-
spatial composition of microbial communities in
specific FISH probe (α-Proteobacteria) and general FISH
environmental samples. CLSM applications to study
probe (domain Bacteria) on wheat roots first
microbial populations in rhizosphere samples have
demonstrated the potential of combining probes of
become numerous during the last decade or so, as seen
different specificity. More recently, Kutter et al., (2005)
laser
Plant Sciences Feed Vol 1 Issue 4
47
scanning microscopy (CLMS)
Confocal
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. used FISH to demonstrate different colonization
nodulation in living alfalfa roots. To specifically monitor
patterns by the pathogens Salmonella enterica and
growth-active Pseudomonas cells, Ramos et al., (2000)
Listeria spp. on barley roots. Watt et al., (2006) used
followed the colonization pattern of an unstable GFP
FISH for quantitative studies of bacteria colonizing
mutant of P. putida on young barley roots.
wheat roots; Pseudomonas and filamentous bacteria were found to comprise 10% and 4%, respectively, of the total rhizosphere community. Other Gram-negative bacteria monitored recently by FISH have been Rhizobium sp. on rape (Santaella et al., 2008) and Methylobacterium suomiense on rice and tomato (Poonguzhali et al., 2008).
Another approach for detection of single cells in the rhizosphere is the use of reporter genes in specific bacteria, also referred to as reporter bacteria, whole cell biosensors, bacterial bioreporters or monitor strains. Such cells are equipped with reporter genes that encode a product, which is easily assayed in fluorescence microscopy and related to metabolic activity or specific
Recent advances to further develop the FISH
gene expression of the host cell. Reporter bacteria
technology have been to target mRNA (rather than
respond to the bioavailable fraction of compounds in
rRNA)
their surroundings.
monitoring
expression
of
toluene
monooxygenase gene (tom) in P. putida in wheat rhizoplane samples (Wu et al., 2008a). At present, the first application of FISH to target cellular DNA monitoring occurrence of nitrite reductase gene (nirK) in indigenous denitrifier populations in environmental samples has been reported (Pratscher et al., 2009). This development is very promising for studying both occurrence (e.g. phylogeny, density, colonization pattern) and metabolic activity (gene expression) of
groups. The first group represents the non-specific reporters, which carry e.g. lux reporter genes under control of a constitutive promoter. Frequently these reporters are tagged with a complete luxCDABE cassette encoding the luciferase as well as genes involved in production of substrate for the enzyme. These bacteria will emit constant light when supplied with oxygen and energy. To track a specific bacterial
rhizosphere bacteria. REPORTER
Reporter bacteria can be classified into three
BACTERIA
AND
REPORTER
GENE
TECHNOLOGY
inoculant in the rhizosphere, the Green Fluorescent Protein
(GFP) technology in
combination with
fluorescence microscopy has had greatest importance for plant microbe interaction studies. The principle of a
the rhizosphere is insertion of fluorescence marker
non-specific reporter strain is illustrated in Figure 1.
genes in specific bacteria under study. Suitable designs
The second group of semi-specific reporters is based on
of plasmid or transposon vectors for insertion, driving
expression of reporter genes in response to stress-full
promoters and a panel of colour variants have allowed
stimuli even when the actual stress factor is unknown.
for bioluminescence
gene) or Green
Finally, the third group of specific reporters may
Fluorescence Protein (e.g. gfp gene) tagging in several
respond to presence of specific compounds or elements
bacteria and microfungi from soil and rhizosphere. The
(e.g. Jaeger et al., 1999) or to their absence (e.g. Koch et
early paper by Gage et al., (1996) described the
al., 2001). Figure 2 illustrates the principle of a specific
construction of Rhizobium meliloti GFP mutants and
reporter. Semi-specific stress reporters may be used
showed detailed CLSM images of their growth and
where bacterial physiology under adverse conditions
behaviour during the early stages of infection and
(e.g. exposure to high temperature or osmolarity,
(e.g. lux
Plant Sciences Feed Vol 1 Issue 4
48 48
An important approach to track single bacteria in
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. drought, or presence of reactive oxygen species) is
STUDIES OF THE RHIZOSPHERE ENVIRONMENT
controlled by global regulatory circuits (regulons) of
USING REPORTER ORGANISMS
gene expression.
Carbon,
nitrogen,
phosphorous
and
oxygen
availability Responses to specific nutrient limitation in soil and rhizosphere represent some of the first applications of reporter bacteria to study specific gene expression in this environment. The approach has been dominated by intensive studies of Pseudomonas sp. strains, most certainly due to the importance in degradation and nutrient cycling by this organism per se, but also due to the potential exploitation of the organism in agricultural biotechnology, e.g. for plant protection, plant growth promotion and bioremediation. Figure 1: In a non-specific bacterial reporter, the luxAB genes encoding bacterial luciferase are inserted behind a constitutive promoter located on the chromosome (or a plasmid) of e.g., Pseudomonas fluorescens. Bioluminescence depends on cellular energy, but also on oxygen and aldehyde substrate (not shown). Toxic compounds or elements such as Cu inhibit generation of cellular energy and thereby the light reaction (Sorensen and Nybroe, 2007).
Root exudates have long been considered to be the major C source supporting growth of root-colonizing bacteria in the rhizosphere of young plants. An advancement has been the use of non-specific lux tagged reporters to detect the actual C-source composition and availability in soil and rhizosphere samples through changes in metabolic activity. Hence, inoculant Pseudomonas sp. (shortly pre-starved for C) responded to both source and concentration of C; wheat root exudates gave a response comparable to that of reducing sugar monomer (glucose), rather than that of common amino acid (glutamate) or carboxylic acid (succinate) components in root exudate (Yeomans et al., 1999). The significance of N and P limitation in soil and rhizosphere has been addressed by studies including bioluminescent Pseudomonas reporter strains (Standing et al., 2003). In bulk soil neither N nor P limitation could be observed in agreement with the above observations of C limitation in this habitat. However, soil amendment
Plant Sciences Feed Vol 1 Issue 4
with barley straw changed the life conditions for the inoculated
Pseudomonas
strain,
encountering
N
limitation when C-rich polymers from the barley residues were degraded (Jensen and Nybroe, 1999; Koch et al., 2001).
49
Figure 2: In a specific bacterial reporter, the luxAB genes are inserted behind an environmentally induced promoter located on the chromosome (or a plasmid) of e.g., Pseudomonas fluorescens. Expression of bioluminescence, e.g. by Cu induction, occurs at concentrations below the level that inhibits the light reaction (Sorensen and Nybroe, 2007).
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. The N reporter strain used by Jensen and Nybroe
degradation
studies
is
that
concerning
the
(1999) reacted towards limitation by both NH4+ and
polychlorinated biphenyls (PCB). This group of much-
common amino acids (e.g. glutamate), and further work
attended pollutants is degraded by several soil bacteria,
should address if specific, reduced N components in
including typical degrading genera such as Ralstonia,
exudates may regulate Pseudomonas spp. growth in the
Burkholderia and Pseudomonas. The initial degradation
rhizosphere.
step is mediated by a biphenyl dioxygenase, encoded by
The study by Jaeger et al., (1999) illustrates
the bphA gene and Layton et al., (1998) constructed an
another advantage by reporter studies, namely that
R. eutropha ENV307 strain harboring the reporter
precise information on the spatial distribution of C and
plasmid pUTK60 with an insert of orf0regulatory-
N compounds can be obtained. These authors made a
bphA1 (including promoter) linked to promoterless lux.
Erwinia herbicola tryptophan-reporter strain with a
The detection limit was approx. 1μM for both
fusion between the aatl gene encoding a tryptophane
monochlorinated
aminotransferase and a inaZ ice nucleation reporter. In
polychlorinated biphenyls (Arachlor 1,242 mixture),
the rhizosphere of an annual grass (Avena barbata), the
but the linear range appeared narrow (approx. 1–
reporter showed significant induction in older root segments with lateral root formation, but not at the root
(2-CB,
3-CB
and
4CB)
and
10μM). By comparison, Cell-cell interactions in the rhizosphere
tip. Oxygen availability in the soil environment is of
Some of the most fascinating applications of
fundamental importance to expression of several
reporter bacteria have addressed the complicated plant
distinguishing traits in Pseudomonas spp., notably
bacterial, fungal-bacterial or even bacterial cell-cell
denitrification but also a number of redox-regulated
interactions in soil systems, as examplified in the
traits like fluorescent siderophore and HCN production.
following. The first well-known example is that of plant
Pollutant aromatics and their degradation
signals (flavonoid compounds) controlling early stages
containing
of the legume-Rhizobium symbiosis via activation of the
bioluminescence or gfp fusions in the functional
bacteral nod genes; an early Rhizobium reporter based
degradation genes have been presented. A first example
on a nodC-lacZ fusion was constructed by Bolanos
to illustrate the progress being made to detect
Vasquez and Warner (1997) to study activation by six
occurrence and degradation of specific chlorinated
different flavonoids from host plants (bean). Further,
aromatics,
2,4-
the identification of new, specific environmental signals
dichlorophenoxyacetic acid (2,4-D) and the degrading
regulating bacterial growth and activity in the
soil bacterium, Cupriavidus necator JMP134 (formerly R.
rhizosphere has become feasible with reporter
eutropha
techniques that can sort out activated gene promoters
Several
specific
involves
JMP134).
This
reporters
the
strain,
herbicide
harboring
the
degradation plasmid pJP4, has become the model of 2,4-
under in vivo conditions.
D degradation; two modules of tfd genes, Rregulatory-
There has also been research using Pseudomonas
DIICIIEIIFII and Tregulatory-CIDIEIFI, are both involved
reporter bacteria to study gene-regulating signals of
in the degradation steps from ring cleavage of 2,4-
importance for interaction with plant-pathogenic
dichlorocatechol to formation of a product entering the
microfungi, including both Oomycota and “true”
TCA cycle.
microfungi. In a study, Lee and Cooksey (2000) found
assessed with reporter bacteria in soil and rhizosphere
that hyphal colonization of Phytophthora parasitica by a P. putida
Plant Sciences Feed Vol 1 Issue 4
50 50
A second example of pollutant compounds being
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. reporter strain led to activated promoters
adopted routinely in microbial ecology in the 1990’s
controlling ABC transporter proteins. The recent report
when the Polymerase Chain Reaction (PCR) and other
of de Werra et al., (2008) demonstrates that GFP-based
DNA-based characterization methods became available.
reporter fusions to the phlA and prnA genes essential
The rapid interest for these methods requiring the DNA
for production of antifungal compounds in P. fluorescens
(or RNA) to be directly extracted from the environment
CHA0 can be constructed to monitor biocontrol gene
was related to their capacity to overcome biases in
expression of this strain in the rhizosphere.
isolation and in vitro cultivation. The new discipline
Finally, a role of bacterial cell-cell communication
opened new possibilities in the search for knowledge
including cell density-dependent gene regulation
within the “black box” of soil microbiology. Despite the
(quorum sensing) in a large number of bacteria has also
introduction of other biases (Wintzingerode et al.,
been indicated in rhizosphere systems. Steidle et al.,
1997), the new DNA- and PCR based approaches have
(2001) constructed AHL-sensitive reporters with GFP in
provided completely new insight into the life of
P. putida and were able to demonstrate that the
microorganisms in their natural environment.
indigenous bacterial community colonizing tomato
Extraction of nucleic acids from complex systems
roots in natural soil produces N-acyl-L-homoserine
Nucleic acid
extraction
from environmental
lactone (AHL) molecules, serving as bacterial cell-cell
samples is the basis for a range of methods used in
communication
molecular
signals;
interestingly,
the
AHL
microbial
ecology.
For
complex
production appeared to be more common among plant-
environments such as bulk soil and rhizosphere two
associated than among soilborne Pseudomonas spp.
approaches have been developed for extracting nucleic
COMMUNITY
STUDIES
IN
RHIZOSPHERE
BY
acids. The first is direct extraction of the nucleic acids
NUCLEOTIDE AND PCR BASED METHODS
after in situ cell lysis which is then followed by DNA
Molecular analysis of microbial communities
purification (Ogram et al., 1987). In the second
Despite improvements in cultivation techniques to
approach the cell fraction is first separated from soil
study the microbial community composition in complex
particles before the cells are lysed and nucleic acids
environmental samples, the organisms in culture
purified (Courtois et al., 2001). Both approaches have
represent only a minor fraction of the microorganisms
advantages and disadvantages related to DNA yields,
occurring in situ as estimated by e.g. DNA re association
DNA purity for molecular purposes, and the ever
(Torsvik et al., 1990). Culturing of microorganisms is
questioned representation of the entire microbial
hampered by difficulties in reproducing natural,
diversity.
ecological niches in the laboratory media. Furthermore,
The direct DNA extraction method
symbiotic relationships might be of crucial importance
The direct DNA extraction method assumes
for community function and are often impossible to
complete in situ lysis of all microorganisms present in
maintain in the cultivation process. Hence, there is a
the sample. The disruption of the microbial cell wall
need
to
must lead to the release of all nucleic acids from all
accompany the cultivation-dependent methods to
bacteria, theoretically independently of the cell wall
obtain a deeper insight into the structure and function
sensitivity to lysis treatments, the location of bacteria in
of indigenous microbial communities.
microstructures and their interactions with soil
for
cultivation-independent
techniques
particles. Purification of the extracted DNA solution is
study the indigenous microbial community were
the second step of the direct DNA extraction methods in
Plant Sciences Feed Vol 1 Issue 4
51
Cultivation-independent molecular approaches to
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. which the aim is to get the best compromise between
environmental research. As mentioned previously 16S
purification efficiency and DNA loss. The direct DNA
rRNA has the advantage of being found in many copies,
extraction method generally provides the highest DNA
and has been the easiest gene transcript to detect. The
yields but some applications, such as construction of
ubiquitous presence of RNases and the very short half-
metagenomic libraries, require the extraction of large
life of RNA (especially mRNA) have made studies based
DNA frag-ments that are rarely obtained by direct
on RNA from environmental samples very difficult. For
extraction methods.
these reasons, the bacterial extraction procedure
The bacteria extraction method
described above is normally not applied when mRNA is
An alternative approach to the direct DNA
the target nucleic acid. Alternatively, quick-freezing
extraction method was developed for the recovery of
samples in liquid nitrogen, direct extraction, pre-
highly purified and large bacterial DNAs essential for
treatment of all solutions with the RNase inhibitor
preparing metagenomic DNA to detect complete gene
DEPC and baking all glassware as well as keeping all
clusters and biosynthetic pathways. The bacteria
samples on ice during the extraction procedure, is
extraction method is based on the initial separation of
advantageous.
bacteria from the soil particles prior to cell lysis and
Based on recent methods for extraction RNA,
DNA purification with gentle treatments to preserve
investigations on the diazotrophic community in the
DNA integrity. Protocols have gradually been improved
rhizosphere of different rice cultivars (Knauth et al.,
by optimizing each of the following sequential steps:
2005) and Spartina alterniflora (Brown et al., 2003) by
dispersion of soil particles, separation of the cells from
detecting nifH mRNA, as well as investigations on the
soil particles by centrifugation and/or buoyant density,
denitrifying community detecting the nirK and nirS
lysis
gene transcripts (Sharma et al., 2005), have been
of extracted
Approaches
cells
combining
and DNA purification. centrifugation-based
cell
performed.
Recently, high-resolution studies on
separation from soil particles and in-plug lysis and
transformation dynamics of the functional gene tfdA
pulsed field gel electrophoresis (PFGE) after bacteria
(Nicolaisen et al., 2008; Bælum et al., 2008) in natural
are embedded in the agarose plugs are now recognized
soil have been made. The extraction protocol used co-
as the most efficient method yielding DNA fragments
extracts of both DNA and RNA, which has the advantage
more than 300 kbp in size and providing adequate
that it offers information on both the community
purity for further molecular cloning procedures (Robe
structure in general and on the active subpopulation.
et al., 2003). Alternative methods combining gentle lysis
The protocol also offers the possibility of relating the
treatments with CsCl density purification have
transcript formation over time to the actual population
recovered very pure, 100 kbp long DNA fragments.
density thus obtaining an “activity per DNA unit”.
RNA extraction and purification
PCR BASED ANALYSIS AND COMPARATIVE STUDIES
During the last decade, focus has shifted from merely looking at diversity and community structure
OF DIVERSITY AND FUNCTION Molecular markers The basis of molecular microbial ecology is the
function. One way has been to look at gene expression
molecular markers. Molecular markers can be genes or
during different environmental fluctuations. Studies on
gene transcripts that can be identified in a complex pool
environmental RNA have mainly focused on 16S rRNA,
of nucleic acids providing information on the group of
and only slowly are studies on mRNA emerging in
organisms harbouring these genes. Information based
Plant Sciences Feed Vol 1 Issue 4
52 52
towards linking community structure to community
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. on DNA and RNA can answer questions related to the
Another approach is to focus on a functional group
population structure of a specific environment such as:
within the microbial community independent of the
Is a particular gene present in this population? What is
phylogenetic relationship. Functional genes encoding
the phylogenetic composition of this community? Are
enzymes central for specific metabolic processes found
particular genes expressed? How is the community
in the rhizosphere have been investigated in this
composition changed after perturbation of the
environment e.g., amoA genes from ammonia oxidizing
environment? What are the spatial or temporal
bacteria and archaea (Chen et al., 2008), nir genes from
differences in a particular habitat? Different levels of
denitrifying bacteria and nif genes from nitrogen fixing
phylogenetic resolution or different functional groups
bacteria (Babic et al., 2008).
of organisms can be detected based on the choice of a
In recent years, RNA has thus been more often
marker. Information based on the small subunit (SSU)
targeted for information on the active fraction of the
rRNA (16S rRNA of prokayotes or 18S rRNA of
population, as transcript formation is believed to follow
eukaryotes) reveals the phylogenetic relationship
metabolic activity. The SSU rRNA has the advantage of
between the organisms from where the DNA or RNA
being present in large amounts in the cells, and thereby
arose (Woese et al., 1990). Over the last decade,
being the easiest of the RNA species to detect in
sequencing of SSU rRNA from uncultured organisms
complex samples. However, microorganisms living in
has led to the development of databases (e.g. Genbank:
the soil environment are often starved, and starved cells
http://www.ncbi.nlm.nih.gov/Genbank/index.html or
have been shown to maintain their ribosomes longer.
Ribosomal Database Project: http://rdp.cme.msu.edu/)
Hence, using 16S rRNA to describe the active
and it has been verified that only a small fraction of the
populations in a soil/rhizosphere environment might
soil microorganism diversity is known. Furthermore,
be questioned. An alternative approach is to study
new genera have been proposed solely on the basis of
mRNA of functional genes, which is believed to have a
SSU rRNA sequences form environmental samples, thus
much shorter half-live than 16S rRNA.
having no representatives in culture (Fieseler et al.,
Analysing specific subpopulations based on the pool of nucleic acids directly is often not possible as
2004).
most detection systems are not sensitive enough to
marker of bacteria is the housekeeping gene rpoB,
detect the often very low amount of a specific gene in
coding for the RNA polymerase beta-subunit. This gene
the sample. By PCR, fragments of specific marker genes
is only found in one copy in all bacteria investigated,
are amplified to reach a concentration that can be
giving some advantages over the often multicopy SSU
detected by several downstream applications discussed
rRNA gene when using genetic fingerprinting for
below. The application can be divided into qualitative
diversity indexes. In addition, the rpoB gene has thus
(describing diversity overall or active), or quantitative
been
phylogenetic
(describing abundance of gene copies or gene
information for diversity studies (Case et al., 2007). The
transcripts) in the environment. To date the PCR assay
rpoB approach has been used to study Paenibacillus
has, despite its limitations, enhanced our knowledge on
spp. in the rhizosphere of sorghum under different
the soil and rhizosphere community dramatically, and
nitrogen regimes (Coelho et al., 2007); however, the
will probably do so for many years to come. For studies
advantage of 16S rRNA for phylogenetic analysis, still
on RNA, a conversion of RNA into complementary DNA
lies in the huge ribosomal database available.
(cDNA) is necessary prior to PCR amplification. This is
shown
to
contain
enough
Plant Sciences Feed Vol 1 Issue 4
53
An alternative to 16S rRNA as a phylogenetic
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. done in the reverse transcription (RT) assay and the
and are selected for further discussion here. The three
joint protocol for reverse transcription and subsequent
techniques are based on three different separation
PCR amplification is referred to as RT-PCR. The reverse
technologies.
transcription can be performed with specific primers
DGGE is based on different melting behaviour of
targeting a specific transcript or it can be performed
double-stranded DNA due to sequential differences in a
using random primers converting all RNA to cDNA. The
denaturing gradient during electrophoresis (Muyzer et
high sensitivity and the ability to analyse a higher
al., 1993). T-RFLP separates fragments based on length
number of samples in a shorter time makes the RT-PCR
of the terminal fragments obtained due to differences in
based detection of RNA from environmental samples
restriction endonuclease sites (as in RFLP and ARDRA)
the most promising, despite the biases introduced.
(Liu et al., 1997). Finally, SSCP separates fragments
Clone libraries and fingerprinting
based on different mobility of single stranded DNA in
Sequencing of clone libraries based on PCR-
non denaturing gels (Schwieger and Tebbe, 1998). It
amplified genes obtained from environmental samples
has been shown that the results obtained based on the
offers the highest phylogenetic resolution and has led to
three methods reveal the same clustering of the
the recognition of the impressive diversity of
microbial members when used on the same soil sample,
prokaryotes. Most obtained sequences are deposited in
and the three fingerprinting methods seem equally
databases like the Ribosomal Database Project (RDP)
suited for analysing differences in community patterns
and GenBank, and can be used for comparative studies
due to physico-chemical and biological differences
on prokaryotic diversity in the environment and to infer
between the sites of investigation (Smalla et al., 2007).
phylogenetic relationships between the organisms
Advantages of the DGGE and SSCP approaches are the
detected. Cloning followed by sequencing of specific
possibility of isolating specific genetic elements for
conserved genes is of great importance in research on
subsequent sequencing, whereas this is not possible
indigenous populations.
when T-RFLP is used, omitting the opportunity to fully
Fingerprinting techniques based on PCR amplicons
identify peaks of interest. On the other hand T-RFLP is
have a high potential for screening multiple samples for
suitable for high-throughput analyses, despite the need
differences in the genetic diversity of a group of genes.
for a restriction digest step. All three methods have
Fingerprinting techniques reveal information on the
been successfully applied in rhizosphere research.
genetic diversity of the sample, but only rarely indicate
DGGE is the most extensively used fingerprinting
phylogenetic relationship of the detectedfragments.
method to study structural diversity in the rhizosphere
Applying
combining
and has been used to investigate a diverse range of
fingerprinting with sequencing (with or without a
scientific questions related to rhizosphere microbiology
cloning step in between) of selected fragments can
e.g. dynamics of methanogenic archaeal communities in
improve the data analysis and add information on
Japanese paddy soils (Watanabe et al., 2007),
community structure.
community structure of Pseudomonas spp. in relation to
a
polyphasic
approach
the antagonistic potential in the rhizosphere (Costa et
terminal restriction fragment length polymorphism
al., 2007) and effects of elevated CO2 concentrations on
(TRFLP) and single-strand conformation polymorphism
the structural diversity of microorganisms in a
(SSCP) of DNA and RNA are currently the fingerprinting
grassland (Drissner et al., 2007).
techniques most often applied in rhizosphere studies,
Plant Sciences Feed Vol 1 Issue 4
54 54
Denaturing gradient gel electrophoresis (DGGE),
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. Quantitative PCR
DNA- and RNA-based Stable Isotope Probing (SIP)
Most studies based on PCR are still qualitative, and
Among other applications related to the direct
only recently has quantitative PCR (and RT-PCR) based
recovery of nucleic acids from the environment are
methods been a routine in molecular microbial ecology.
those which can provide a direct access to relate
Traditional PCR rely on end-point detection of the
function of a microbial community and identification of
amplicons. Due to chemical and physical properties
the bacteria that account for it. The establishment of
included in the PCR reaction, the end-point detection of
such a relationship still remaining a great challenge in
the product is not quantitative. However, several
microbial ecology. Stable-isotope probing (SIP) was
quantitative PCR methods have been introduced and
introduced to microbial ecology by Radajewski et al.,
used in rhizosphere research including MPN-PCR
(2000) and has been used to characterize growing
(Rosado et al., 1996), competitive PCR (Mauchline et al., 2002), and most recently real-time PCR (Mavrodi et al., 2007). Real-time PCR is presented here as the most promising
method
to
quantify
genes
from
environmental samples. By real-time PCR, amplicon formation is monitored in real time using fluorescence techniques, where fluorescence detected is proportional to amplicon formation. This enables the detection of product over the full amplification curve, and inhibition of the reaction e.g., by inhibitory substances co-extracted with the nucleic acids, can easily be identified. Another major
microorganisms in environmental samples or to determine those which have the genetic potential of metabolizing a labeled substrate. For instance, the DNAstable isotope probing (DNA-SIP) technique that combines isotopic 13C tracer incorporation into the DNA or RNA and molecular approaches as described above can help to identify soil and rhizosphere bacterial populations that are actively involved in the carbon cycle. The principle of these techniques is to provide soil bacteria with
13C-labelled
material, e.g. cellulose
produced by Acetobacter xylinus as reported by El Zahar
advantage of real-time PCR is the applicability over a
Haichar et al., (2007) and photosynthates released by
wide range of initial DNA concentration of target gene
plants grown under artificial atmospheres (Ostle et al.,
extracted from the environment (Heid et al., 1996).
2003) before extracting total DNA from the soil and
Several detection formats have been developed for the
separating the 13C -labelled (heavy) and unlabelled
real-time PCR assay, SYBR green detection and TaqMan
(light) DNA fractions by ultracentrifugation.
probes being the most commonly used in molecular
METAGENOMIC DNA LIBRARIES
microbial ecology. SYBR green detection is not
Modern highthroughput technology has made
sequence specific as SYBR green binds all dsDNA in the
it possible to not only look for specific genes but look at
minor groove. Upon binding to dsDNA, fluorescence
the full genomic information present in a soil sample,
enhances about 100-fold in comparison with unbound
called the metagenome (Rondon et al., 2000). With such
SYBR green, and it is therefore well suited for detection
bacteria extraction methods clones containing soil DNA
of product formation. Being a non-specific dye, accurate
inserts ranging in size from 40 kb to 50 kb can be
quantification of nucleic acids is dependent on high
routinely obtained. Even without robot facilities
specificity of the PCR reaction per se. The TaqMan
metagenomic libraries of more than 150,000 clones can
probe format relies on the specific binding of a
be prepared and handled for molecular, biological or
hybridization probe to the target sequence and the
chemical
cleavage of the probe by the endonuclease activity of
equivalent of 1,500 E. coli genomes such libraries are
Plant Sciences Feed Vol 1 Issue 4
Although
representing
an
only a partial representation of the initial genetic
55
the Taq DNA polymerase.
screenings.
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. diversity of the bulk or rhizospheric soil samples tested when at least two millions of such clones would be necessary to statistically consider that all the initial genomes have been included at least once in the library. Several screening strategies of metagenomic DNA libraries were developed with direct recombinant expression as a first alternative to detect metabolic activities (Figure 3). The usual option consists in using the high transformation efficiency of E. coli for creating metagenomic libraries although this is certainly not the best heterologous host to express genes from soil bacteria (and even if expression efficiency can be artificially increased). For instance high throughput anti-infective assays can be run by spotting the E. coli recombinant clones on bacteria- or fungi-seeded agar plates subsequently analyzed to detect growth inhibition halos around the positive clones while other
Figure 3: Schematic representation of the metagenomic approach. A sample collected in the environment including soil (1) is submitted to treatments to extract and purify bacterial DNA (2). Several possibilities exist to deal with the extracted metagenomic DNA (3). One of the most common approaches is to use PCR amplification (4) as a fist step before products are
bioassays are achieved by preparing an extract from
cloned and sequences. Bacterial diversity can thus be
each E. coli recombinant clone to analyze its potential
estimated with the help of phylogenetic trees constructed
activity versus a set of chemical reference compounds
from 16S rDNA or other gene sequences (5). Another
and of negative controls.
approach is based on the use of fingerprinting techniques such
A second alternative relies on a molecular screening of the library requiring the spotting of colonies on high density membranes or chips before hybridizing membranes with conserved domains of targeted genes, the main difficulty consisting in
as T-RFLP, DGGE and SSCP that produce fragments to characterize bacterial diversity in the analyzed sample (6). A second approach involves the direct cloning of extracted DNA (7) in a domesticated host such as Escherichia coli to produce metagenomic DNA libraries (8). These libraries can be screened according to 3 methods, including a molecular
designing the probe(s) encompassing the unknown
screening with clone DNA transferred to membranes for
genetic diversity (Demaneche et al., 2009).
hybridization (9), or a chemical screening by analyzing the
Advances like the 454 pyrosequencing technology
culture supernatant of metagenomic clones to detect
has opened new perspectives in sequencing the
compounds specifically produced by insert DNA (10). A last
metagenome of environmental DNA (Demaneche et al.,
method is to spread the clones on a selective medium to detect
2009). By pyrosequencing, adapters are added to each DNA fragment obtained from the environmental sample, and subsequently these fragments are added to a bead—one fragment per bead. This technology has
those for which insert gene expression permits clone growth (11). Screening by these two last methods is based on expression of insert genes and transfer of recombinant plasmids, fosmids or BACs in alternative bacterial hosts can increase the recovery of genes of interest. The last approach which is called to become very popular in the next future is to
sequencing, but has the disadvantage that it currently
sequence directly the metagenomic DNA with the help of new
only read 100 bases per run.
sequencing technologies such as 454.
Plant Sciences Feed Vol 1 Issue 4
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dropped the time and costs constraints of DNA
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. DNA microarrays and whole-community studies of
hybridization signals with rhizosphere amplified DNA
diversity and function
compared to the bulk soil DNA, suggesting that this
The use of extraction-purification kits that permit
group should be considered a rhizospheric group, while
several soil samples to be processed simultaneously has
Acidobacteria, Bacteroidetes, Verrucomicrobia, and
contributed
DNA-based
Planctomycetes related probes indicated that these
approaches to study bacteria in their natural
bacteria should not be considered as rhizospheric.
environment. However, the huge biodiversity and
Microarray technologies were also developed to detect
functional capabilities of bacteria in these samples
bacterial gene families that encode key enzymes
cannot be monitored without the corresponding high-
involved in the ecosystem functioning (Wagner et al.,
throughput technologies requested to analyze DNA
2007).
solutions.
TRANSCRIPTOME ANALYSIS
to the success of the
DNA
microarrays
can
fulfil
these
requirements by hybridizing in a single step soil
In addition to study the composition and genetic
amplified and labeled DNA targets to thousands of DNA
capabilities of the bacterial community in their natural
probes targeting genes of interest immobilized on solid
environment, DNA microarrays can also be used to
surfaces, the hybridization signal of each probe being
determe the transcript level of all the genes in a given
subsequently and simultaneously recorded with a
gene pool. For this purpose the total transcript of a
detector (Wagner et al., 2007; Huyghe et al., 2008).
sample is obtained by total mRNA extraction. This been
mRNA is then converted into DNA (cDNA) by reverse
developed depending on the target genes, the so-called
transcription, labelled (usually with a fluorescent dye)
Phylo-Chips (or phylogenetic oligonucleotide arrays)
and hybridized to microarrays containing the DNA
referring to the detection of the ubiquitous ribosomal
genes whose expression is to be analyzed. The level of
genes to detect and identify theoretically any
the signal is proportional to the level of transcript
Two
main
microarray
systems have
There are two types of commercially available
and the functional gene microarrays for detection and
microarrays. One type contains the open reading frames
analysis of specific protein-encoding (functional) genes
(ORF) of the relevant genome, synthesized by PCR and
(Wagner et al., 2007). Targeting 16S or 23S rRNA genes,
spotted on a solid microarray (or macroarray) matrix.
Phylo-Chips have begun to be used for the detection and
The other type is the oligonucleotide microarrays, in
identification of microbial strains, species, genera or
which the probes are short sequences designed to
higher taxa (depending on the design of the probe)
match parts of the sequence of known or predicted
including the rhizosphere environment. For instance,
ORFs. These microarrays are produced by printing
the high-throughput analysis potential of Phylo-Chips
short oligonucleotide sequences designed to represent
detected a significant rhizosphere effect when a 170
a single gene or family of gene splice-variants by
probe microarray was used to compare the maize
synthesizing this sequence directly onto the array
rhizosphere and the bulk soil (Sanguin et al., 2006). This
surface instead of depositing intact sequences. The size
study showed that taxonomic groups such as
of the sequences varies, according to the producer (i.e.,
Sphingomonas spp., Rhizobiaceae, and Actinobacteria
60-mer probes such as the Agilent design or 25-mer
were identified in both rhizosphere and bulk soil with
probes such as produced by Affymetrix). Although the
strong hybridization signals, indicating no specific
longer probes are more specific to individual target
habitat preference for these groups. In contrast,
genes, shorter probes may be spotted in higher density
Agrobacterium spp. targeting probes yielded stronger
across the array and the cost of their production is
Plant Sciences Feed Vol 1 Issue 4
57
microorganism in the soil sample (Desantis et al., 2007);
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. lower. Microarrays based on oligonucleotides enable
and one with a fluorescence emission wavelength of
the quantization of transcripts from non-coding regions
670 nm (corresponding to the red part of the light
of the DNA. This is important for the analysis of small
spectrum). The two differentially labelled cDNA
RNAs (Gottesman et al., 2006) and regulatory regions of
samples are mixed and hybridized to a single
the transcripts. For specific purposes, or when
microarray that is then scanned in a microarray scanner
commercial microarrays are not available, it is possible
to visualize fluorescence of the two fluorophores
to produce microarrays “in house”. In these microarrays
(Figure 4). These microarrays provide data on the
(spotted microarrays) the probes are oligonucleotides,
transcription level of the whole genome under two
cDNA or small fragments of PCR products that
conditions, as well as a comparison of the two.
correspond to the expected transcripts. These arrays
PROTEOME ANALYSIS
may be easily customized for each experiment, and are especially
useful
for
expression
profiling
of
environmental samples.
Gene expression can also be analysed at the level of translation—the final level of gene expression. Most of the experiments to analyze the composition of proteomes use
high
resolution
two-dimensional
polyacrylamide gel electrophoresis (2D gels) coupled to identification of proteins by mass spectrometry. Twodimensional gel electrophoresis makes it possible to resolve complex mixtures of cellular proteins. In this method proteins are separated by their isoelectric point and molecular mass. Proteins are extracted from microorganisms, tissues or other samples and are separated by their isoelectric point, on a gradient of pH (1st dimension). They
are then
separated by
electrophoresis on SDS-polyacrylamide (SDS-PAGE) according to their molecular mass (Neidhardt and van Bogelen, 2000). The result of this procedure is a gel with proteins spread out on its surface. These proteins are then detected by staining (usually silver or Coomassie stains). The staining provides data on Figure 4: Two-color microarray approach comparing transcriptomes in cultures grown at 32°C and 42°C, respectively.
approximate protein amounts, which is adequate for most purposes. Two-dimensional gel electrophoresis has become a very powerful tool to resolve proteins, which are the final stage of gene expression (Volker and Hecker,
prepared from two samples to be compared (e.g.,
2005). It should be noted that there exist alternative
bacteria at two temperatures) that are labeled with two
technologies, such as multi-dimensional-HPLC coupled
different fluorophores (Shalon, 1998), often one which
mass spectrometry and protein-chip techniques, for
has a fluorescence emission wavelength of 570 nm
proteomic
(corresponding to the green part of the light spectrum),
analysis
without
the
use
of
gel
electrophoresis. However, these methods have not yet
Plant Sciences Feed Vol 1 Issue 4
58 58
Two-Color microarrays are hybridized with cDNA
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. been extensively used in the study of gene expression in
data indicate that post-translational modifications
environmental samples (Wolff et al., 2007). Comparison
appear to be more common than expected. As an
of two samples, and analysis of proteome expression
example, when a theoretical proteome was compared to
induced by environmental and physiological changes, is
the actual proteome of A. tumefaciens, there were many
performed by overlaying two gels and quantifying the
proteins that deviated from the expected (theoretical)
difference between them. The analysis can be
molecular weight (MW) or pI as measured by their
performed by one of several image analysis programs.
vertical and horizontal migration distances, respectively
More accurate results are obtained by running the two
(Rosen et al., 2004). These proteins were clearly
samples on one gel. This sort of experiment is often
subjected to post-translational modifications, which
used to determine the effect of an environmental
changed their pI and/or molecular weight. Proteomic
change on the composition of the proteome. For this
studies can be carried out and provide solid data even
type of experiment, the culture is metabolically labelled
on organisms whose genomes are not yet sequenced.
(with L-[35S]methionine or [14C]amino acid mixture)
Future challenges for proteomics thus include the
when exposed to the stress. The protein synthesis
development of efficient technologies for the study of
pattern is obtained by autoradiography can be directly
membrane proteins and the improvement of automatic
compared with the protein level pattern. Because the
software based analysis to overcome problems with
total proteins and the newly synthesized proteins (i.e.,
incompletely separated (overlapping) spots and weak
stress-induced proteins) are on the same gel there is no
spots.
need for matching several gels and proteins that belong to different stimulons or regulons can be identified (Gottesman et al., 2006). The radioactive proteome and the total proteome (obtained by staining) can be differentially stained in the computer, and the relative gene expression is obtained by analysis of the colors. Figure 5 shows the vegetative (computer stained in green color) and heat shock (computer stained in red color) proteomes of Agrobacterium tumefaciens, as compared to the control in which the label was added to unstressed cultures. Using this method it is possible to define the protein signatures of microorganisms in response to a variety of environmental conditions and to identify environmentally induced regulons. Proteomic analysis provides information about the final levels of cellular proteins, thus measuring the level of the end product of gene expression. In addition, it provides information
Figure 5: Proteomic approach analyzing a temperature upshift. A bacterial culture growing at 32°C was incubated 20 min in the presence of 35S methionine at 32°C (left side) or 42°C (right side). The proteomes were analyzed by two dimensional electrophoresis. Each gel was autoradiographed and the image was computer-stained in red. The gel was then silverstained and the image was computer-stained in green. The overlays of radioactive and silver-stained images are shown.
Another
newly
developed
approach
is
“proteogenomics” - which combines metagenomics
previously assumed that post-translational modification
data with mass spectrometry based proteomics. It
plays only a minor role in prokaryotes. However, recent
uses proteomics approaches to verify coding regions
Plant Sciences Feed Vol 1 Issue 4
59
about posttranslational modifications. It has been
B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. of metagenomic sequences and quantify their activity.
REFERENCES
This technology is based on the ability to directly
[1]
measure expressed
peptides arising proteins
by
from
proteolysis of
high-throughput
liquid
chromatography tandem mass spectrometry-based proteomics. This new technology has not yet been extensively used in rhizosphere studies, but is expected to provide powerful tools in this area of research (Wilmes et al., 2008a, 2008b). CONCLUSION The new discipline of molecular microbial ecology was often seen as the “panacea”, without considering that other limitations than those related to cultivation, could strongly bias results on the actual extent of bacterial diversity and functions. However, when researchers are aware of these biases and limitations, molecular microbial ecology still provides new insight not previously obtained by the cultivation dependent approaches. In addition, better cultivation dependent approaches are developing fast, and today research moves towards a polyphasic methods approach including both lines of research in order to gain most knowledge with the least bias. Furthermore, in reaching the goal of understanding the complex microbial communities in the rhizosphere, the systematic identification and quantitation of all metabolites in the rhizosphere, termed metabolomics, can be of major importance as an additional approach to include in the studies. The present availability of a large number of whole genome sequences constitutes a leap forward in the understanding of microbial communities in the rhizosphere, as well as well as the genetic composition of their individual constituents. The
availability
of
sequences
together
with
bioinformatics tools will enable us to translate the sequences into functions.
Plant Sciences Feed Vol 1 Issue 4
60 60
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