Microbes Environ. Vol. 22, No.

2, 93–105, 2007
http://wwwsoc.nii.ac.jp/jsme2/

Minireview

Microbial Community Analysis of the Phytosphere Using

Culture-Independent Methodologies

ASAMI SAITO1, SEISHI IKEDA1*,†, HIROSHI EZURA2, and KIWAMU MINAMISAWA1

1 Graduate School of Life Sciences, Tohoku University, 2–1–1 Katahira, Aoba-ku, Sendai, Miyagi 980–8577,
Japan
2 Gene Research Center, University of Tsukuba, 1–1–1 Tennoudai, Tsukuba, Ibaraki 305–8572, Japan

(Received March 9, 2007—Accepted March 27, 2007)

The phytosphere is an attractive habitat for microorganisms due to an abundance of nutrients and relative envi-
ronmental stability. The microorganisms that occupy this habitat assist in the uptake of nutrients from soils and
can exert considerable influence upon the overall health of the plant. Recent technical advances in environmental
microbiology have enabled the tracing and assessment of these microorganisms using rapid and simple molecu-
lar techniques without any culture-dependent bias. We herein review the current status of these modern molecu-
lar techniques in the study of plant-associated microbes, and summarize the issues relevant to the phytosphere
from the aspect of both basic and applied science.

Key words: Community analysis, microbial diversity, plant-microbe interaction, phytosphere

Conventional culture-dependent methodologies have pro-

Introduction
vided useful information for evaluating microbial diversity
The phytosphere is a most attractive habitat for microor- in various environments including the phytosphere. How-
ganisms due to the availability of many nutrients and its ever, these conventional methods are limited by strong
environmental stability. Conceptually, this unique environ- inherent biases caused by the medium selected and the cul-
ment consists of three main habitats for plant-associated ture conditions. Moreover, a significant disadvantage of
microbes: the phyllosphere, the endosphere and the rhizo- these techniques is the inability to culture most of the
sphere. Each of these three habitats provides a considerably microbes in nature1). In contrast, recent technical advances
diverse physical, chemical, and biological environment, and in environmental microbiology have enabled the evaluation
as a consequence can support a wide range of microbial of microbial diversity using rapid, simple, and less biased
groups. The microorganisms in these habitats assist plants culture-independent molecular techniques54). Culture-inde-
in the uptake of several vital nutrients from the soil, such as pendent methodologies have now revealed that the majority
phosphorous, potassium and nitrogen9,13,30,108,110), and some of plant-associated microbes have not yet been cultured in
of these organisms can exert considerable influence upon the laboratory2,51,117). These studies have thus provided vital
the overall health of the host plant26,27,39,80,101,104). clues regarding the abundance and spatial distribution of
microbial groups in the phytosphere. However, the relation-
* Corresponding author. E-mail address: sikeda@nbrc.nite.go.jp;
ships between plants and phytosphere microbes are still
Tel.: +81–438–20–5764; Fax: +81–438–52–2314.
† Present address: NITE Biological Resource Center (NBRC), largely unknown as the phytosphere contains a broad spec-
National Institute of Technology and Evaluation (NITE), 2–5–8 trum of microbes in terms of their degree of interaction with
Kazusakamatari, Kisarazu, Chiba 292–0818, Japan. the host plant. These range from neutral microorganisms
94 SAITO et al.

with no obvious effects upon the host plant to pathogens sampling methods, depending on the target microbial com-
and mutualistic symbionts with deleterious and beneficial munity.
effects, respectively.
The use of currently available molecular techniques will (1) Phyllosphere
clearly facilitate studies of plant-associated microbiology in The phyllosphere is defined as a microbial habitat mainly
both basic and applied research areas. However, these associated with the surface of the leaf. Prior to sampling of
molecular techniques also have several limitations in terms the phyllosphere, it must be considered that the composition
of their application to microbial community analyses. In the of the microbial communities therein can be influenced by
present review, we first discuss technical issues in the appli- several factors including plant growth stage, leaf aging,
cation of culture-independent methodologies to the study of pathogen infections, local temperature and humidity, and
plant-associated microbes mainly focusing on bacteria and the accidental presence of transient saprophytes. Thus, the
fungi. These issues include sampling, DNA extraction, PCR phyllosphere is a relatively variable and harsh environment
amplification, and DNA fingerprinting for a microbial com- compared to the endosphere and rhizosphere.
munity analysis of the phytosphere. Subsequently, we sum- Two sampling methods should be considered when eval-
marize the current status of the application of microbial uating microbial communities in the phyllosphere. The first
community analyses in the fields of plant-associated micro- of these involves the extraction of microbial cells from the
biology. surfaces of leaves50,81,117), either by simply washing tissue
with a specific buffer50), or in combination with
sonication117). This method provides relatively pure environ-
Plant managements for community analyses mental DNA in terms of both chemical and biological quali-
Microbial community analyses of the phytosphere are ties for microbial community analyses. This is due to lower
often performed in a controlled environment such as a levels of contamination from plant debris during the extrac-
greenhouse or a test field. In such cases, however, parti- tion. This technique can also isolate high molecular weight
cular care needs to be taken with regards to plant manage- DNA suitable for the construction of a large insert library
ment practices both before and during the analysis. Differ- such as a Bacterial Artificial Chromosome (BAC) library,
ences in management procedures, such as the application when used in combination with chemical and enzymatic cell
of fertilizers and pesticides, have been shown to cause lysis procedures50). It must be considered, however, that the
structural changes to plant-associated microbial commu- efficiency with which cells are recovered from the surfaces
nities32,70,97,106,116). In addition, structural changes to micro- of plant tissues may depend on factors such as plant/tissue
bial communities can be caused by differences in the levels type, the age of the tissue, and differences in the microbial
of mycorrhizal fungi present in the rhizosphere69,71). Hence, groups present. An example of this has been shown in anal-
efforts must be made to maintain the homogeneity of the yses of epiphytic microbes present on leaves and seeds,
test soils as much as possible. Plant genotypic variation at which are considerably affected by the large variability
the intraspecies level has also been reported to have a con- among samples81,117). Dent et al.15) have previously
siderable impact on the composition of microbial employed a culture enrichment method involving seed
communities57), indicating the importance of considering the imbibition for 16 hours, followed by tissue fractionation
genetic purity of plant materials for community analyses of prior to DNA extraction. However, this procedure should be
the phytosphere. Moreover, the growth stage of plants has avoided when evaluating microbial diversity, because it can
been found to strongly influence microbial community change the ratio of individual species in a population, par-
structures64,68,103). These considerations may not apply in the ticularly in the case of bacteria.
case of a microbial community analysis using natural sam- The second method involves the direct use of plant tis-
pling sites for ecological studies, but could provide better sues for extracting DNA by bead-beating or the use of a
insight for the interpretation of the results. mortar and pestle43,81). In this case, samples contain both
epiphytic communities (a microcosm in the phylloplane or
spermoplane) and endophytic communities (the endophyllo-
Sampling of phytosphere materials sphere or endospermosphere). This procedure is simple and
The microbial habitats present in the phytosphere com- faster than the first method. However, there is likely to be
prise diverse physical, chemical, and biological environ- contamination by excess plant DNA which could affect sub-
ments, and these differences require the use of a variety of sequent molecular analyses by PCR. Although the appropri-
Microbial Community Analysis of the Phytosphere 95

ate choice of primers and reaction parameters could circum-

vent or minimize these potential biases, the amount of (3) Rhizosphere
microbial biomass in the phyllosphere or in seeds is gener- The rhizosphere was initially defined as the soil environ-
ally extremely small relative to the total plant material. Con- ment directly under the influence of the living roots of the
sequently, the analysis of communities in these tissues is host plant, but more recently the term has come to include
often strongly biased by the presence of plant DNA and both roots (endorhizosphere and rhizoplane) and root-asso-
may in some cases be impossible to conduct81). The sam- ciated soil (ectorhizosphere or rhizosphere soil)
pling procedures for the phyllosphere have been success- environments84). Although the effects of the rhizosphere on
fully applied to the culture-independent analysis of the the diversity of soil microbes can usually be observed by
microbial diversity in seeds42,43,81). community analyses (Fig. 1), a precise physical definition
of rhizosphere soils is extremely difficult as the degree of
(2) Endosphere influence by the roots on neighboring soil environments can
The endosphere is defined as the microbial habitat inside be affected by several factors including plant species, plant
of plant tissues. The sampling methods currently employed aging, and soil properties13,14,59,70,72,82,103,115,116). For practical
for analyzing the endosphere can also be divided into two purposes, two sampling methods are usually employed
main types. The first of these employs a procedure for steril- when analyzing the communities of the rhizosphere. The
izing the surface of stem tissues using chemical reagents first consists of the recovery of adherent soil by agitation
and/or flaming in order to eliminate contamination from
epiphytic microbial DNA2,88). This approach has been
widely used for the isolation of endophytes (microbes
inhabiting the endosphere)78), as well as for analyzing
microbial communities in the endosphere2). The surface ste-
rility of tissues is generally assessed by placing the steril-
ized tissues on appropriate growth medium. However,
although this procedure is effective in eliminating potential
contaminants during the isolation of endophytes based upon
conventional culture methods, it may be insufficient to
definitively establish the absence of microbial DNA derived
from the dead cells of epiphytes (microbes inhabiting the
phyllosphere on the surface of plant tissue).
Discrepancies between the results of community analyses
of the endosphere using culturable and non-culturable meth-
ods may be partially due to the persistence of bacterial DNA
on dead cells from the plant surface. Hence, some reports
have proposed the aseptic peeling of the surfaces of the
plant tissues prior to DNA extraction89,98). However, as
reported by Reiter and Sessitsch90), aseptic peeling is
impractical for some plant materials. In this case, the analy-
sis of bacterial communities using cultivation-independent
methods should be defined as a plant-associated community
analysis that includes both epiphytes and endophytes.
The second method for analyzing communities of the
endosphere utilizes a procedure for extracting bacterial cells
from the insides of plant tissues29,90). This comprises a Fig. 1. RISA Profiles of the bacterial communities in maize rhizo-
mechanical disruption of the bacterial cells and has been sphere. M, molecular size markers; lane B and R indicate bulk
shown to be effective in minimizing contamination by plant and rhizosphere soil samples, respectively. Triplicate results for
each sample are shown. The leftmost numbers indicate marker
DNA prior to DNA isolation29,90). Although this method is
fragment lengths. Fewer amplicons of increased intensity in the
more laborious than the first, it facilitates a less biased anal- rhizosphere soil samples are considered to indicate rhizosphere
ysis of endophytic microbial diversity. effects.
96 SAITO et al.

(shaking) of roots that have been decomposed carefully recovery of DNA/RNA from the phytosphere has yet to
from the ground, either in air or water19,62,92). In the case of a become routine, and still requires refinement of the extrac-
bacterial community analysis, the resulting rhizosphere soil tion and purification conditions due to the extreme diversity
fraction can then be subjected to cell extraction prior to of the physical, chemical, and biological properties of plant
DNA preparation. materials. It is also well recognized that the methods
The advantage of this method is that it can minimize con- employed for extracting DNA or RNA can themselves bias
tamination by plant tissues. However, the quality and quan- the results of microbial community analyses in terms of
tity of the recovered rhizosphere soils can also be greatly both qualitative and quantitative interpretations of data66).
affected by the handling procedures and by other factors Moreover, whereas RNA-based examinations of microbial
such as the properties of both the soil and root systems. In communities may provide a better indication of the natu-
addition, some researchers may use procedures for extract- rally occurring profiles, the changes in expression levels of
ing bacterial cells from rhizosphere soils prior to DNA RNA may be too sensitive to environmental stress, such as
extraction that have various modifications such as bead that during the extraction process, to obtain reliable data and
shaking37) and sonication73), making it difficult to directly appropriate interpretation of the results.
compare the results. Another concern with this method is In addition, RNA molecules are extremely labile both in
that excess amounts of bulk (non-rhizosphere) soil could be vitro and in vivo, and therefore may not be an appropriate
carried over into the rhizosphere fractions and thereby mask marker for evaluating certain environmental impacts. Con-
the impact of the rhizosphere upon the soil microbial com- sequently, soil DNA has been analyzed in the most recently
munity structures. reported studies of microbial communities in the phyto-
A second established method for sampling rhizosphere sphere due to a lack of reliable methodologies for RNA
soil is the direct use of root systems with tightly adherent extraction. We therefore mainly focus on the relevant con-
soil for DNA extraction, without the separation of plant tis- siderations when extracting DNA from the phytosphere in
sues. This procedure recovers microbial DNA from the this review. The details of some of the DNA extraction pro-
rhizoplane region and retains less bulk soil compared with cedures that can be used for recalcitrant environmental sam-
the first method. As a result, the microbial DNA that is sam- ples, such as rhizosphere soils, are referred to our recent
pled could be expected to better reflect the microbial diver- review47).
sity in the rhizosphere. However, there are also some poten- The extraction of DNA usually involves three steps; cell
tial problems with this procedure. First, there may be lysis, extraction of nucleic acids, and subsequent purifica-
contamination by excess plant DNA, in which case the tion steps. For the efficient lysis of microbial cells, bead-
appropriate choice of PCR primers and careful technique beating is often employed as the initial step of the extraction
will be needed to minimize any potential bias. Second, sev- procedure. However, subsequent extraction/purification
eral root tips are usually collected for DNA extraction, since steps have tended to vary among different laboratories.
it is practically impossible to extract microbial DNA Hence, until recently no established DNA extraction
directly from the entire root systems of most field crops. method had been reported for analyzing the communities of
This may also bias the results due to the presence of a shift the phyllosphere and seeds. For analysis of the endosphere,
in the microbial community structures from the root to the bead-beating treatments of ground tissue have been con-
basal area within the same root system116). Third, consider- ducted, followed by the standard phenol-chloroform extrac-
able spatial variability in the vertical distribution of soil tion and/or CTAB extraction. Importantly however, these
microorganisms exists due to the presence of a surface gra- extraction/purification procedures were originally devel-
dient for several environmental factors, including oxygen oped for extracting plant DNA, and were not actually
availability and various nutrients. In order to control for intended for use with microbial DNA. Hence, these meth-
these variations in a given sampling site, it is advisable that ods may not be adequate for the efficient extraction of DNA
multiple samples be collected rather than the individual from microbial cells in the phytosphere.
sample volume be increased. We have shown in our recent study that a soil DNA
extraction method could be adapted for the simple and rapid
preparation of environmental microbial DNA directly from
DNA extraction diverse biological materials, including plants and related
To date, several reports have described effective ways to agronomic products44). More recently, a soil DNA extrac-
extract nucleic acid from the phytosphere. However, the tion method has been employed in several studies for ana-
Microbial Community Analysis of the Phytosphere 97

Table 1. The influence of the extraction method on the quality of commercial soil DNA kits such as the FastDNA SPIN Kit
plant-associated DNA for Soil (QBioGene/MP Biochemicals, Inc., Solon, OH,
OD260/OD230b OD260/OD280c
USA) and UltraClean Soil DNA Isolation Kit (Mo Bio Lab-
Samplea oratories, Inc., Carlsbad, CA, USA). Because phytosphere
Soil kitd Plant kite Soil kit Plant kit
samples often contain inhibitors of various enzymatic reac-
Leaf 0.9±0.7f 0.02±0.002 1.6±0.4 2.0±0.05 tions including PCR, the success of these analyses is highly
Stem 0.6±0.4 0.02±0.005 1.6±0.02 1.9±0.2 dependent upon the purity of the microbial DNA and thus
Root 0.6±0.3 0.02±0.0003 1.7±0.1 1.8±0.2 upon the effectiveness of the method of extraction
a Tissues were subjected to bead-beating in a DNA extraction buffer employed.
prior to the extraction. The impact of the two DNA extraction methods on analy-
b The index for polysaccharide contamination.
c The index for protein contamination.
ses of the microbial communities of the phytosphere has
d FastDNA SPIN Kit for soil. e FastDNA SPIN Kit. f Mean±S.D. been evaluated in our laboratory. The DNA prepared with a
soil DNA extraction kit clearly showed a high ratio of
OD260/OD230, which indicates less polysaccharide contami-
nation, as compared to the DNA prepared with a plant DNA
extraction kit (Table 1). On the other hand, the two methods
gave similar values for the OD260/OD280 ratio as an index of
protein contamination (Table 1). More importantly, these
results reflected the differences of fingerprinting profiles as
shown in Figure 2. These results clearly indicate the impor-
tance of the DNA extraction method employed, and of the
quality of the environmental DNA when analyzing the
microbial communities in the phytoshpere.

PCR amplification
PCR amplification of ribosomal RNA regions has been
extensively used to study microbial diversity as this meth-
odology takes advantage of the accumulation of such
sequences in the public databases83). In general, both small
and large subunit rRNA genes and their intergenic spacer
regions are utilized for primer design in microbial diversity
studies as they are present in all organisms (Table 2). In
most of the current reports of microbial communities, DNA
fingerprinting techniques have been widely employed in
combination with PCR amplification of ribosomal RNA
regions47). However, it has been estimated that microbial
Fig. 2. RISA Profiles of the bacterial communities in the soybean community analyses that employ culture-independent meth-
phytosphere. Lane M, molecular size markers; lane S and P indi- odologies can detect only 1–2% of the total microbe popula-
cate DNA samples prepared from each tissue using a soil DNA tions in a complex environmental sample such as soil67).
extraction kit and a plant DNA extraction kit, respectively. Tripli-
This is mainly due to the low resolution power of currently
cate results for leaf, stem and root tissues are shown. The leftmost
numbers indicate marker fragment lengths. The influences of the available fingerprinting techniques against the diversity of
DNA extraction methods on the results of the microbial commu- microbial communities in nature109).
nity analysis are indicated by the differences in the number and In cases where certain microbes are not easily detectable
intensity of the DNA bands between the S and P samples. by the use of standard universal primer sets, taxon-specific
primers have been shown to be sometimes effective (Table
lyzing microbial communities in the phyllosphere and 2)2,36,56). In the phytosphere, several microbial groups, such
seeds28,32,42,43). Similarly, the most recent analyses of com- as Burkholderia, Pseudomonas, and Actinomycetes, are
munities in the rhizosphere have successfully employed known to be important community members, and specific
98 SAITO et al.

Table 2. PCR primers employed to analyze the microbial communities of the phytosphere

Community or Organism Target region Method Primer (Forward/Reverse)a References

Seedb
Bacteria 16S rRNA DGGE 341F-GC/534R and Anti-chloroF/534R, 15)
Bacteria ITSc RISA ITSF/ITSReub 43)
Fungi 18S rRNA DGGE NS1/NS8 and Eukaryote specific primers 15)
Fungi ITS RISA 1406f/3126T 43)
Phyllosphere
Bacteria 16S rRNA DGGE F341/R534 81)
Bacteria 16S rRNA DGGE 968-1401 38)
Bacteria 16S rRNA DGGE primer1/primer2 50)
Bacterioplankton 16S rRNA DGGE PRBA338f/PRUN518r 117)
Endosphere
Bacteria 16S rRNA DGGE F341/R534 81)
Bacteria 16S rRNA DGGE F968-GC/R1378 2)
Bacteria 16S rRNA DGGE P388f/P518r 97)
Bacteria 16S rRNA T-RFLP 243f/1492r 10)
Bacteria 16S rRNA T-RFLP 799F/1520R 87)
Bacteria 16S rRNA T-RFLP 799f/pH 41)
Bacteria 16S rRNA T-RFLP 8f/926r 89)
α-proteobacteria 16S rRNA DGGE Fα-U/R1378 and F968-GC/R1378 2)
β-proteobacteria 16S rRNA DGGE Fβ-2/R1378 and F968-GC/R1378 2)
Pseudomonas bacteria 16S rRNA DGGE 8f-GC/PSMGx 91)
Actinomycetes 16S rRNA DGGE F243-R518GC 98)
Actinomycetes 16S rRNA T-RFLP 8f/518r 98)
Fungi 18S rRNA DGGE NS1/FR1-GC 28)
Fungi 18S rRNA DGGE EF4f/NS3r 97)
Rhizosphere
Archaea 16S rRNA DGGE A46f/A1117r and A340f-GC/A533 105)
Bacteria 16S rRNA DGGE F341-GC/R518 19)
Bacteria 16S rRNA DGGE F984-GC/R1378 37)
Bacteria 16S rRNA DGGE PRBA338f/PRUN518r 116)
Bacteria 16S rRNA T-RFLP 27 Forward/1525 Reverse 52)
Bacteria 16S rRNA T-RFLP 8-27f/1507-1492r 20)
Bacteria ITS RISA 1405F/23R 3)
Bacteria ITS RISA ITSF/ITSReub 45)
Bacteria 16S rRNA SSCP 133FN6F/248R5P 102)
Bacteria 16S rRNA SSCP Com1/Com2-Ph 96)
α-proteobacteria 16S rRNA DGGE F203α/R1494 and F984GC/R1378 36)
β-proteobacteria 16S rRNA DGGE F948β/R1494 and F984GC/R1378 36)
Bacteroidetes 16S rRNA DGGE C319/907R and 341FGC/907R 32)
Burkholderia 16S rRNA DGGE Burk3-GC/BurkR 93)
Actinobacteria 16S rRNA DGGE F243/R1494 and F984GC/R1378 36)
Actinomycete 16S rRNA DGGE F243/R513-GC 37)
Methylotrophs 16S rRNA DGGE 142F/533R 23)
Methylotrophs 16S rRNA DGGE 197F/533R 23)
planctomycetes 16S rRNA T-RFLP PLA-40F/1492R 16)
Pseudomonades 16S rRNA DGGE F311Ps/1459Ps 76)
Streptomycete 16S rRNA DGGE StrepB/Strep and E341f-GC/534r 105)
Eukaryote 18S rRNA DGGE NS1-GC/NS2 76)
Fungi 18S rRNA DGGE NS0/EF3 and NS1/FR1-GC 12)
Fungi 18S rRNA DGGE NS1/FR1-GC 31)
Fungi 18S rRNA DGGE NS1/NS2+10-GC 107)
Fungi 18S rRNA DGGE NS1-GC/NS2 72)
Fungi ITS RISA 1406f/3127T 45)
Fungi ITS RISA 2234C/3126T 46)
Fungi ITS T-RFLP ITS 1F/ITS 4 56)
VA fungid 18S rRNA DGGE AM1/NS31-GC 60)
Ascomycetes ITS T-RFLP ITS 1F/ITS 4A 56)
Ascomycetes ITS DGGE ITS5/ITS4A-GC 113)
Basidiomycetes ITS T-RFLP ITS 1/ITS 4B 56)
a Two primer sets are shown for nested PCR. b Seed-associated microbial community.
c Internal transcribed spacer region between the small subunit rRNA and large subunit rRNA genes. d Vesicular-arbuscular mycorrhizal fungi.
Microbial Community Analysis of the Phytosphere 99

primer sets for these microbes have been develop- PCR amplifications in our laboratory for the analysis of
ed36,76,93,98,105). By using group-specific primers, Costa et diverse biological materials including phytosphere44,47).
al.13) have also revealed that the extent of “rhizosphere Other additives such as GC-Melt (Clontech, Palo Alto, CA,
effects” is largely dependent on the plant species, as well as USA) may also be of great assistance in PCR amplifications
the microbial groups under examination. Although a series of target sequences containing a high GC content, as is the
of PCR primer sets are available as “universal primers”, one case for Actinomycetes.
should keep in mind that none of the presently available While the microbial community in a phytosphere sample
primers will amplify all sequences from the corresponding may contain a high level of diversity, it may also consist of
eukaryotic, bacterial, or archaeal domains. taxonomically similar groups of microbes. In the latter case,
In the case of microbial community analyses in the endo- mis-priming during PCR may become a major problem and
sphere, it has been reported that the major portion of the result in the formation of chimeras during amplification. In
clone library for a (partial) 16S rRNA gene fragment, which order to minimize this, two strategies are usually employed
was amplified by using universal primers for eubacteria, for analyzing microbial communities. These are the use of a
often contains plant organelle-derived 16S rRNA “hot start program”74) and a “touch down program”19), and
sequences98). In order to circumvent this problem, Chelius these conditions can also be used in combination.
and Triplett8) have reported the use of a primer (799f) that
was designed for the specific amplification of bacterial 16S
rRNA gene sequences directly from root tissues. Although
Fingerprinting techniques
the successful application of this primer has been shown in There are four principal fingerprinting techniques that
several studies41,86), it has further been reported that this have been widely applied to microbial community analyses
primer set may have underestimated the diversity of micro- of the phytosphere. These are denaturing gradient gel elec-
bial communities based on a comparison of the T-RFLP trophoresis (DGGE), single strand conformation polymor-
profiles with another set of primers41). In addition, a bias of phism (SSCP), terminal restriction fragment length poly-
this primer for proteobacteria has been described by Reiter morphism (T-RFLP), and ribosomal intergenic spacer
and Sessitsch90). Recently, Rasche et al.87) have shown that analysis (RISA). The principles, advantages, and disadvan-
their 16S rRNA gene libraries, generated using the 799f tages of these techniques have been described in recent
primer, contain large numbers of clones assigned as chloro- reviews47,55). Therefore, we have herein focused on the cur-
plast sequences, indicating that the specificity of this primer rent status of the application of these techniques to micro-
is also dependent upon the genotypes of the plant organelle. bial community analyses of the phytosphere in the present
Caution should therefore be taken when microbial commu- review.
nity analyses are conducted with this primer set.
While the assessment of bacterial diversity is less prob-
lematic due to the availability of universal primers for bac-
RNA-based community analysis
terial domains, fungal community analysis suffers from the DNA-based community analyses do not necessarily
effects of co-amplification of DNA from other eukaryotic reflect the metabolic activity or prove the viability of the
organisms such as plants, algae, and nematodes61). Although corresponding organisms, due to the presence of dead cells
there are several studies that have reported attempts to or extracellular DNA in the environment under study.
resolve this problem56,58), the specificity of these primer sets Hence, a RNA-based community analysis is more suitable
is still not sufficient, especially when applied to fungal com- for elucidating the metabolically active members of a bacte-
munity analyses of the phytosphere. rial population, since the amount of rRNA can generally
Due to the technical limitations in the purification of be correlated with the growth activity of bacteria114). To
environmental DNA samples, it is not feasible to expect the date, several reports have described the results of RNA-
complete elimination of all potential contaminants from the based community analyses, and distinct differences have
phytosphere materials. In order to overcome the potential been observed between DNA- and RNA-based anal-
inhibition of PCR by such contaminants, and to perform sta- yses18,57,90,91,100). In these studies, the RNA-derived community
ble PCR amplifications, a series of special additives are profiles were found to be less complex than their DNA-
often incorporated into the amplification mixtures. Among derived counterparts. As a consequence, one of the most
these, we recommend bovine serum albumin (BSA), as it is recent achievements in phytosphere microbiology is the
relatively inexpensive and has helped to generate stable application of stable isotope probing (SIP) in combination
100 SAITO et al.

with RNA-based fingerprinting85). Plant roots release 1– modified rhizobia on rhizosphere microbial communities
25% of their net photosynthetic metabolites as soluble and have been extensively examined28,92,96,107,111,112). Despite a
insoluble compounds into the rhizosphere75). By taking number of environmental assessments of upland transgenic
advantage of this fact, microbial community analyses have crops, no transgenic rice has so far been investigated for the
revealed the flow of stable isotopically labeled carbon from effects upon microbial communities in the phytosphere.
the atmosphere into microbes in the rhizosphere65,85). However, an examination of the possible impact of trans-
genic rice on microbial communities may be very important
environmentally, since altering the genotypes of rice plants
Microbial community analysis in phytosphere by
may either increase or decrease global CH4 emissions from
culture-independent methodologies
rice fields63,77,79). These microbial community analyses of
(1) Assessment of biotic and abiotic environmental phytospheres could thus allow us to examine the possible
factors impact of GMOs on the environment more comprehen-
The use of culture-independent methodologies to analyze sively, and minimize the environmental risk in the utiliza-
microbial communities has potential when assessing the tion of GMOs in open fields such as the culturing of trans-
environmental impact of biotic and abiotic factors on plant- genic plants or releasing of genetically modified microbes
associated microbial communities in both natural and agri- as a biological control agent or a plant growth promoter.
cultural settings. Because of concerns about global warm- Microbial community analyses of the phytosphere have
ing, the impact of elevated CO2 levels upon the rhizosphere also begun to provide new insights into the relationships
was examined as they could possibly affect microbial com- between the incidence of disease and microbial diversity.
munity structures through alterations in the carbon flow Disease symptoms are not always visible on infected plants,
from photosynthetic activities49,56). Similarly, the rice rhizo- and specific diseases can remain latent for long
sphere has also been subjected to microbial community periods6,11,35,94). Because the economic losses associated with
analyses due to growing concerns regarding methane emis- latent infections are considerable for some plants, including
sion from rice fields. Lu and Conrad65) applied a RNA- various tree species, due to their long period of cultivation,
based community analysis to the identification of methano- microbial community analyses may be useful as diagnostic
genic Archaea in the rice rhizosphere, in combination with tests to guarantee pathogen-free conditions, at least at the
stable isotope-probing techniques, and have shown that a time of planting25). In addition, these community analyses
methanogenic archaea group is mainly responsible for CH4 provide an opportunity to screen for potential new antago-
production in rice field soil. nistic microbes which may be useful biological control
Methane-oxidizing bacteria (MOB) have also been recog- agents for plant pathogens in the phytosphere. In this
nized as an important microbial group in the reduction of regard, McSpadden-Gardener and Weller73) have studied
methane emissions from rice agriculture. Therefore, evalua- microbial community structures in disease suppressive soil
tions of the community structures of this group of bacteria to survey candidate antagonistic microbes responsible for
have been conducted by several groups23,40), and shown that this suppression. More recently, Reiter and Sessitsch90) have
the population size and activity of MOB in the rhizosphere reported that the high tolerance of a variety of potato against
were mainly affected by plant growth stages. Dohrmann and common scab may be at least partly due to its ability to host
Tebbe17) have also shown that ozone stress has only small some endophytic Streptomycetes.
effects on the structural diversity of the bacterial communi-
ties in the rhizosphere. These findings indicate the useful- (2) Diversity of microbial functional genes
ness of the culture-independent community analyses of the One of the main problems associated with analyzing
phytosphere to global environmental assessments. microbial communities is the difficulty in interpreting any
Environmental risk assessments of genetically modified changes of the fingerprinting profiles in terms of their bio-
organisms (GMOs) have become one of the major areas for logical significance due to the use of rRNA gene regions.
the application of culture-independent microbial community This is because most microbes in nature are unculturable
analyses. The impact of transgenic plants on the microbial and their functionality can therefore not be predicted accu-
community in the phytosphere has been examined, and was rately based on the analysis of their rRNA gene regions,
found to be negligible in comparison with natural varia- unless the functionality can be reflected in their phyloge-
tions such as plant growth stages and growth condi- netic locations. In order to circumvent this problem, several
tions4,7,21,24,28,33,86,95,98). Similarly, the impact of genetically studies of the diversity of microbial functional genes in the
Microbial Community Analysis of the Phytosphere 101

Table 3. PCR primers employed for the molecular analysis of functional microbial genes in the phytosphere

Community or
Organism
Target gene Method Primer (Forward/Reverse)a References

Endosphere
Bacteria nifH Sequencing nifH(for A)/nifH(rev) and nifH(forB)/nifH(rev) 88)
Rhizosphere
Bacteria Chitinase T-RFLP GA1F/GA1R 45)
Bacteria mmoX Sequencing 534f/1393r 40)
Bacteria mxaF DGGE 1003F/1562R 23)
Bacteria nifH Sequencing nH17K-F/nH139P-R 22)
Bacteria nifH Sequencing nifH(for A)/nifH(rev) and nifH(forB)/nifH(rev) 34)
Bacteria nifH T-RFLP Zehr-nifHf/Zehr-nifHr 106)
Bacteria nifH T-RFLP nifH-F/nifH-R 45)
Bacteria nirK DGGE nirK1F/nirK5R 99)
Bacteria nirS DGGE nirS1F/nirS6R 99)
Bacteria phlD DGGE DGGE292forCG/6DGGE618rev 5)
Bacteria phlD DGGE DGGE292forCG/DGGE618rev 5)
Bacteria pmoA DGGE A189-GC/A682 40)
Bacteria pmoA Sequencing f1003/r1561 40)
Bacteria pmoA T-RFLP A189/A682 40)
a Two primer sets are shown for nested PCR.

phytosphere have been reported (Table 3). The diversity of the composition of the chitinolytic bacterial community45,48).
diazotroph communities in the phytosphere has been stud- Bergsma-Vlami et al.5) have successfully assessed the
ied extensively based on molecular analyses of the nifH genetic diversity of antagonistic Pseudomonas species
genes, due to the importance of this process during crop based on their root colonization ability in the rhizosphere by
production22,34,88,106). Recently, Knauth et al.57) have also using DGGE to target a biosynthetic gene for an antibiotic.
shown that intraspecies genotypic variations among plants The results of these reports reemphasize the usefulness of
have significant influences on the diversity of the root-asso- culture-independent methodologies for analyzing the func-
ciated nifH genes, and suggested that the genetic factors in tionality of microbial communities in the phytosphere.
rice plants that stimulate N2 fixation by diazotrophs can be
identified.
Microbiological denitrification also has become an
Conclusions
important area of research due to its influence on the loss of Whereas a series of culture-independent methodologies
fixed nitrogen in different environments, and on the accu- are now available for analyzing microbial diversity and
mulation of nitric oxide and nitrous oxide which contribute functionality, caution should be taken when performing
to global warming and the destruction of the stratospheric such analyses for the phytosphere, as described in the
ozone layer. Similarly, Sharma et al.99) have examined the present review. Culture-independent community analyses
molecular diversity of the nirK genes, which encode a key will not only be useful for analyzing the roles of uncultured
enzyme in the denitrification process, in the rhizosphere of microbes, but also provide new insights into the known ben-
grain legumes. eficial or deleterious microbes in the phytosphere. The
In the phytosphere, several microbial groups are consid- application of culture-independent methodologies will thus
ered important for protection against plant disease. Among facilitate a better understanding of plant-microbe interac-
these, the chitinolytic bacteria are thought to be important tions across a broad spectrum of microbiological research.
for disease control in the phytosphere53,118). Recently, we
have examined the molecular diversity of root-associated
bacterial chitinase genes, and have shown the significant
Acknowledgements
influence of the rhizosphere as well as plant genotypes on This work was supported in part by a Grant-in-Aid for
102 SAITO et al.

Scientific Research on Priority Areas (‘Comparative 14) De Ridder-Duine, A.S., G.A. Kowalchuk, P.J.A. Klein Gunnew-
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