Vol. 24, No. 4, 2011 / 395
MPMI Vol. 24, No. 4, 2011, pp. 395–407. doi:10.1094/MPMI-05-10-0115. © 2011 The American Phytopathological Society
Effects of Jasmonic Acid, Ethylene,
and Salicylic Acid Signaling
on the Rhizosphere Bacterial Community
of Arabidopsis thaliana
Rogier F. Doornbos,1 Bart P. J. Geraats,2 Eiko E. Kuramae,3 L. C. Van Loon,1 and Peter A. H. M. Bakker1
1Plant-Microbe Interactions, Institute of Environmental Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The
Netherlands; 2Seed Technology, Nunhems Netherlands B.V., Voort 6, 6083 AC, Nunhem, The Netherlands; 3Microbial Ecology,
Netherlands Institute of Ecology, Droevendaalsesteeg 10, 6708 PB Wageningen, The Netherlands
Submitted 19 May 2010. Accepted 9 December 2010.
Systemically induced resistance is a promising strategy to
control plant diseases, as it affects numerous pathogens.
However, since induced resistance reduces one or both
growth and activity of plant pathogens, the indigenous mi-
croflora may also be affected by an enhanced defensive
state of the plant. The aim of this study was to elucidate
how much the bacterial rhizosphere microflora of Arabi-
dopsis is affected by induced systemic resistance (ISR) or
systemic acquired resistance (SAR). Therefore, the bacterial
microflora of wild-type plants and plants affected in their
defense signaling was compared. Additionally, ISR was
induced by application of methyl jasmonate and SAR by
treatment with salicylic acid or benzothiadiazole. As a com-
parative model, we also used wild type and ethylene-insen-
sitive tobacco. Some of the Arabidopsis genotypes affected
in defense signaling showed altered numbers of culturable
bacteria in their rhizospheres; however, effects were depend-
ent on soil type. Effects of plant genotype on rhizosphere
bacterial community structure could not be related to
plant defense because chemical activation of ISR or SAR
had no significant effects on density and structure of the
rhizosphere bacterial community. These findings support
the notion that control of plant diseases by elicitation of
systemic resistance will not significantly affect the resident
soil bacterial microflora.
The bacterial rhizosphere microflora plays an important role
in plant health. A well-studied phenomenon is the suppression
of soilborne plant diseases by plant root–inhabiting bacteria,
including members of genera Bacillus, Erwinia, Pseudomonas,
Rhizobium, Serratia, and Xanthomonas (Weller 1988; Whipps
2001). Mechanisms of suppression of plant diseases by such
bacteria include competition for substrates, competition for
iron by siderophores, antibiosis, lytic activity, and induced sys-
temic resistance (ISR) (Van Loon and Bakker 2003). Induced
resistance is the state of enhanced defensive capacity devel-
oped by plants when appropriately stimulated (Van Loon et al.
1998; Van Wees et al. 2008; Zehnder et al. 2001). Rhizobacte-
ria-mediated ISR is effective against a wide range of patho-
gens on dicotyledonous plant species, including Arabidopsis,
bean, carnation, eucalyptus, radish, tobacco, and tomato (Bakker
et al. 2007), as well as the monocot rice (De Vleesschauwer et
al. 2008). ISR resembles pathogen-induced systemic acquired
resistance (SAR) in that i) upon challenge inoculation, induced
plants show an enhanced defensive capacity, enabling the plant
to respond faster, more effectively, or both to microbial attack-
ers (Conrath et al. 2002, 2006; Van Wees et al. 2008) and ii)
both are dependent on a functional NPR1 gene (Pieterse and
Van Loon 2004). However, whereas SAR is dependent upon
the plant hormone salicylic acid (SA) and is associated with
the expression of pathogenesis-related (PR) proteins, rhizobac-
teria-mediated ISR in Arabidopsis does not require SA signal-
ing, nor is it associated with the expression of known defense-
related genes (Pieterse et al. 1996, 1998; Van der Ent et al.
2008; Van Wees et al. 1999; Verhagen et al. 2004). Instead,
ISR requires responsiveness to jasmonic acid (JA) and ethyl-
ene (ET); yet, it is not associated with endogenous increases of
these hormones (Pieterse et al. 2000).
In general, pathogens with a necrotrophic lifestyle are re-
sisted by JA- and ET-dependent defenses, whereas SA-depend-
ent defenses are effective against pathogens with a biotrophic
lifestyle (Glazebrook 2005; Thomma et al. 1998). This differ-
ential effectiveness of plant defenses is also displayed by ISR
and SAR (Ton et al. 2002). For example, ISR is effective
against the necrotrophic fungus Alternaria brassicicola,
whereas SAR is not; SAR is effective against the biotrophic
Turnip crinkle virus, whereas ISR is not. However, almost
nothing is known about effects of the augmented defensive
state on the indigenous rhizosphere microflora. A recent study
by Micallef and associates (2009) assessed the rhizobacterial
community structure of eight Arabidopsis accessions. Of
these, the non-ISR–expressing accessions RLD and WS-0
showed a bacterial community structure that tended to differ
from that of the other six, which are ISR-inducible. Such dif-
ferences might be related to differences in plant defensive ca-
The aim of this study was to investigate whether the bacte-
rial rhizosphere microflora is affected by one or both the JA/ET-
or SA-dependent defense-signaling pathways. Two experimental
approaches were used. First, we analyzed the bacterial rhizos-
phere microflora of Arabidopsis thaliana accession Col-0 and
derivatives affected in specific defense signal-transduction
pathways. Second, we activated the JA- and SA-signaling
pathways by exogenous application of the hormones, to study
the effect of activated defenses on the bacterial abundance and
Corresponding author: P. A. H. M Bakker; E-mail: firstname.lastname@example.org;
Telephone: +31 (0)30-2536861; Fax +31 (0)30-2518366.
396 / Molecular Plant-Microbe Interactions
In addition, we used tobacco and its ET-insensitive transfor-
mant, Tetr18 (Knoester et al. 1998; Wilkinson et al. 1997).
Tetr18 plants have reduced resistance against several soilborne
fungi and oomycetes, including species belonging to genera
Fusarium, Thielaviopsis, and Pythium (Geraats et al. 2003;
Knoester et al. 1998). Geraats (2003) suggested that Tetr18
plants differ from nontransformed plants in root characteristics
that influence bacterial community structure in the rhizos-
phere. The Arabidopsis JA-response mutant jar1 and ET-sig-
naling mutant ein2 also have an enhanced susceptibility to dif-
ferent soilborne Pythium isolates (Geraats et al. 2002). More-
over, Arabidopsis genotypes affected in SA signaling display
an enhanced sensitivity to the necrotrophic soilborne oomycete
Phytophthora parasitica, the leaf spot fungus Cercospora nico-
tianae, and the hemibiotrophic bacterial leaf pathogen Pseudo-
monas syringae pv. tomato (Cao et al. 1994; Delaney et al.
1994). Conversely, the constitutive expressor of SA-dependent
defenses cpr1 has increased resistance to the downey mildew
oomycete Hyaloperonospora arabidopsidis as well as to P.
syringae pv. maculicola (Bowling et al. 1994).
In order to obtain representative results, plants were grown
on different soil types: i) a mixture of commercially available
potting soil and sand and ii) a clay soil. Both soils were used
as such or were autoclaved to allow possible species-specific
recolonization from surviving bacteria or the ambient environ-
ment. The rhizosphere microflora, with focus on the total bac-
terial community and Pseudomonas spp., was explored using
two complementary techniques, i.e., culturable-dependent
semiselective plating was used for bacterial quantification and
denaturing gradient–gel electrophoresis (DGGE), a culturable
independent-fingerprinting technique, was used to monitor
possible shifts in microbial community structure.
Abundance of rhizosphere populations
of culturable bacteria and Pseudomonas spp.
The numbers of culturable bacteria and Pseudomonas spp.
in the rhizospheres of Arabidopsis and tobacco plants were
quantified by selective plating. To study a possible role of
defense signaling, mutants affected in either the JA/ET or SA
response were used. Power to detect differences between treat-
ments was 100% for the total bacterial populations and 97%
for numbers of pseudomonads in the nonautoclaved potting
soil, as determined by power analysis. Population densities of
total culturable bacteria in the rhizospheres of the different
Arabidopsis genotypes grown on distinct soils ranged from 2 ×
107 to 1 × 109 colony-forming units (CFU) per gram of rhizos-
phere soil (Fig. 1). Significant differences in bacterial numbers
were only observed for plants grown in untreated potting soil-
sand mixture (Fig. 1A). The JA-response mutant jar1, the ET-
response mutant ein2, and the constitutive SA-producing cpr1
showed significantly lower numbers of culturable bacteria com-
pared with the Col-0 wild type. These differences were not
observed when the potting soil and sand mixture was auto-
claved before use or when plants were grown on nonauto-
claved or autoclaved clay soil (Fig. 1B through D). Numbers
of CFU of Pseudomonas spp. in the rhizosphere were between
5 × 105 and 5 × 107 per gram of root and demonstrated
tendencies similar to total bacterial numbers, except for ein2
(Fig. 2). However, Pseudomonas populations seemed more
sensitive to SA-dependent defenses, as illustrated by a de-
creased abundance in cpr1 and a tendency of increased bac-
terial numbers in the NahG rhizospheres (Fig. 2A and B).
Although differences in bacterial abundance between the geno-
Fig. 1. Number of total culturable bacteria in the rhizosphere (log CFU per gram of root) of Arabidopsis Col-0 and Col-0 derivatives affected in jasmonic
acid- (jar1 and npr1), ethylene- (etr1, ein2 and npr1), or salicylic acid– (cpr1, npr1 and NahG) dependent defense responses. Rhizosphere samples of five
individual plants were dilution plated on 1/10 TSA+ (3 g of tryptic soy broth per liter, 13 g of agar technical per liter, and 100 mg of natamycin per liter). A,
Plants grown on potting soil-sand mixture. B, Plants grown on autoclaved potting soil-sand mixture. C, Plants grown on clay soil. D, Plants grown on
autoclaved clay soil. Different letters indicate significant differences (analysis of variance and Tukey post-hoc test, P < 0.05); error bars represent standard
Vol. 24, No. 4, 2011 / 397
types were not observed when plants were grown on the clay
soil, Pseudomonas population densities were also consistently
lower in the cpr1 rhizosphere (Fig. 2C and D).
The transgenic Tetr18 tobacco contains the mutant etr1 ET
receptor of Arabidopsis, resulting in ET insensitivity. When
grown in either a potting soil-sand mixture or clay soil,
autoclaved or not, no differences were observed in rhizosphere
population densities of total culturable bacteria or Pseudomo-
nas spp. (data not shown). These results are largely compara-
ble to those of the Arabidopsis etr1 and ein2 genotypes.
Composition of total bacterial
and Pseudomonas populations in the rhizosphere
of Arabidopsis defense signaling mutants.
Possible differences in the bacterial rhizosphere community
of the different Arabidopsis genotypes were studied in a cul-
turable-independent manner, using polymerase chain reaction
(PCR)-DGGE with eubacterial- and Pseudomonas spp.–specific
primers. DGGE analysis revealed complex banding patterns
containing 22 to 30 and nine to 18 distinct bands for plants
grown on potting soil and clay soil, respectively. We used re-
dundancy analysis (RDA) to evaluate effects of plant genotype
on the rhizosphere microflora. Ordination plots of the eubacte-
rial data are shown in Figure 3, and those for the Pseudomonas
data are shown in Figure 4.
Analysis of similarity (ANOSIM) showed that the bacterial
rhizosphere microflora was not significantly affected by plant
genotype (Table 1). However, in all soils, the eubacterial
rhizosphere microflora from mutants jar1, etr1, and ein2 con-
sistently clustered away from the Col-0 wild type. Similar re-
sults were obtained for the Pseudomonas spp. community.
Independent of the soil used, the genotypes jar1, etr1, and ein2
clustered separately from the wild type, whereas the other
genotypes showed more variable patterns in the different soils.
Bacterial and Pseudomonas community structure
in the rhizosphere of ET-insensitive tobacco.
The eubacterial and Pseudomonas rhizosphere communities
of wild type and Tetr18 tobacco were not significantly differ-
ent, as assessed by ANOSIM analysis of PCR-DGGE finger-
prints (Table 2). Plants grown in nonautoclaved soil showed
distinct clusters of the eubacterial microflora between the wild
type and the Tetr18 transformant. This effect was less pro-
nounced for the rhizosphere community of plants grown in
autoclaved soil (Fig. 5). The rhizosphere Pseudomonas com-
munity only revealed separate clusters between wild type and
Tetr18 tobacco for plants grown on a nonautoclaved potting
soil and sand mixture (Fig. 6).
Effects of foliar application of methyl jasmonate (MeJA),
SA, and benzothiadiazole (BTH)
on local and systemic VSP2 and PR-1 expression.
In the experiments with the defense-signaling mutants, no
consistent differential effects of plant genotypes on rhizosphere
bacterial communities were observed. Given that defense signal-
ing was not elicited in these experiments, such an experimental
outcome may not be surprising. To investigate if activetion of
JA- and SA-signaling pathways affects the composition of the
rhizosphere microflora, MeJA, SA, or BTH were applied to the
leaves. Application of MeJA activated the JA-dependent defense
response both locally (leaves) and systemically (roots), indicated
by an about 7.5-fold increase in the expression of the JA-respon-
sive marker gene VSP2 (Fig. 7A). Although SA and BTH appli-
cation did not induce expression of VSP2 in the leaves, the ex-
Fig. 2. Number of total culturable Pseudomonas spp. in the rhizosphere (log CFU per gram of root) of Arabidopsis Col-0 and Col-0 derivatives affected in
jasmonic acid- (jar1 and npr1), ethylene- (etr1, ein2 and npr1), or salicylic acid–dependent (cpr1, npr1 and NahG) defense responses. Rhizosphere samples
of five individual plants were dilution plated on KB+. A, Plants grown on potting soi-sand mixture. B, Plants grown on autoclaved potting soil-sand mixture.
C, Plants grown on clay soil. D, Plants grown on autoclaved clay soil. Different letters indicate significant differences (analysis of variance and Tukey post-
hoc test, P < 0.05); error bars represent standard errors.
398 / Molecular Plant-Microbe Interactions
pression of VSP2 in the roots was increased 2.8- and 1.6-fold.
Local expression of the SAR marker gene PR-1 was increased
49-fold in response to SA and 73-fold in response to BTH treat-
ment (Fig. 7B). Foliar application of SA or BTH also resulted in
activation of the corresponding defense-signaling pathway in the
roots, as indicated by a nine- and fourfold induction of PR-1
expression, respecttively. As expected, application of MeJA did
not affect PR-1 expression.
Effect of activated defense signal-transduction pathways
on bacterial abundance.
The abundance of the bacterial and Pseudomonas spp. micro-
flora in the rhizospheres of mock-, MeJA-, SA-, and BTH-
treated plants was quantified by selective plating two weeks
after treatment (Fig. 8). The numbers of CFU of total culturable
bacteria in the rhizosphere of Col-0 revealed no significant influ-
ences of the hormone treatments. Likewise, activation of plant
defenses had no effect on the numbers of total culturable bacte-
ria in the rhizospheres of the mutants npr1 and jar1 nor in trans-
genic NahG plants. Colony counts of Pseudomonas spp. showed
comparable results. However, compared with the mock-treated
control, significantly higher numbers of Pseudomonas spp. were
measured in NahG plants treated with SA or BTH.
Effect of activated plant defense signaling
on bacterial community structure.
Ordination plots derived from DGGE fingerprints of the
bacterial rhizosphere communities were generated by RDA
and revealed that all samples clustered predominantly by plant
genotype (Fig. 9). Induction of defense signaling in the differ-
ent genotypes did not affect rhizobacterial communities, as the
variables mock, MeJA, SA, and BTH are all in the center of
the ordination plot. Pseudomonas-specific DGGE analysis re-
vealed a higher variability among the samples, resulting in less
Fig. 3. Ordination biplots generated by redundancy analysis of eubacterial denaturing gradient–gel electrophoresis fingerprints of the rhizosphere of
Arabidopsis Col-0 and Col-0 derivatives affected in jasmonic acid- (jar1 and npr1), ethylene- (etr1, ein2 and npr1), or salicylic acid–defense (cpr1, npr1 and
NahG) signal-transduction pathways. A, Plants grown on potting soil-sand mixture. B, Plants grown on autoclaved potting soil-sand mixture. C, Plants
grown on clay soil. D, Plants grown on autoclaved clay soil. Crosses represent individual samples, triangles the centroid position of each genotype.
Vol. 24, No. 4, 2011 / 399
distinct clusters compared with the eubacterial analysis. How-
ever, RDA analyses indicated that none of the hormone treat-
ments had a relevant influence on the Pseudomonas commu-
nity structure. Indeed, two-way ANOSIM analysis showed that
eubacterial communities were significantly different with
some overlapping (R = 0.41924) in the rhizospheres of differ-
ent Arabidopsis genotypes, while there were no significant
effects of the hormone treatments (Table 3). For the Pseudo-
monas community, ANOSIM revealed significant differences
in the rhizospheres of different Arabidopsis genotypes, but
also, activation of defense signaling by applying hormones had
no significant impact (Table 3) for this microbial group.
Rhizobacteria-mediated ISR and pathogen-induced SAR can
reduce disease severity and microbial proliferation of a wide
range of plant pathogens (Durrant and Dong 2004; Loake and
Grant 2007; Van Loon 2007; Van Loon et al. 1998; Van Wees et
al. 2008). However, it is not known if augmented plant-defense
responses affect indigenous populations of plant-associated
microorganisms. To elucidate if the bacterial rhizosphere
microflora of Arabidopsis is affected by one or more JA-, ET-,
or SA-dependent defenses, we investigated the abundance and
community structure of the total bacterial microflora as well as
the prevalent Pseudomonas spp. in the rhizosphere of different
Arabidopsis and tobacco genotypes with altered defense
Effect of plant defense signaling on bacterial abundances.
Total bacterial and Pseudomonas spp. population densities
in the rhizospheres of Arabidopsis and tobacco and of deriva-
tives of these plants with altered defense signaling properties
were studied by dilution plating on selective media. Bacterial
Fig. 4. Ordination biplots generated by redundancy analysis of Pseudomonas spp.-specific denaturing gradient–gel electrophoresis fingerprints of the
rhizosphere Arabidopsis Col-0 and Col-0 derivatives affected in jasmonic acid- (jar1 and npr1), ethylene- (etr1, ein2 and npr1), or salicylic acid–defense
(cpr1, npr1 and NahG) signal-transduction pathways. A, Plants grown on potting soil-sand mixture. B, Plants grown on autoclaved potting soil-sand mixture.
C, Plants grown on clay soil. D, Plants grown on autoclaved clay soil. Crosses represent individual samples, triangles the centroid position of each genotype.
400 / Molecular Plant-Microbe Interactions
Table 1. One-way analysis of similarity of denaturing gradient–gel elec-
trophoresis fingerprints of eubacteria and Pseudomonas spp. in the rhizos-
pheres of Arabidopsis Col-0 and Col-0 derivatives affected in jasmonic
acid- (jar1 and npr1), ethylene- (etr1, ein2 and npr1), or salicylic acid-
(cpr1, npr1, and NahG) defense signal-transduction pathwaysa
Potting soil and sand
Autoclaved potting soil and sand
Autoclaved clay soil
a Plants were grown on potting soil and sand mixture, autoclaved potting
soil-sand mixture, clay soil, and autoclaved clay soil.
b R values significant at P < 0.05.
Fig. 5. Ordination biplots generated by principal component analysis of eubacterial denaturing gradient–gel electrophoresis fingerprints of the rhizosphere of
tobacco wild type and its ethylene-insensitive transformant Tetr18. A, Plants grown on potting soil-sand mixture. B, Plants grown on autoclaved potting soil-
sand mixture. C, Plants grown on clay soil. D, Plants grown on autoclaved clay soil. Crosses represent individual samples, triangles the centroid position of
Table 2. One-way analysis of similarity of denaturing gradient–gel elec-
trophoresis fingerprints of eubacteria and Pseudomonas spp. in the rhizos-
phere of tobacco wild type and its ethylene-insensitive transformant Tetr18
Soil Eubacteriab Pseudomonas spp.b
Potting soil and sand
Autoclaved potting soil and sand
Autoclaved clay soil
aPlants were grown on potting soil and sand mixture, autoclaved potting
soil-sand mixture, clay soil, and autoclaved clay soil.
bR values not significant at P < 0.05.
Vol. 24, No. 4, 2011 / 401
population densities in the rhizospheres of the jar1 and ein2
mutants were significantly lower compared with those in the
wild-type rhizosphere when plants were grown in nonauto-
claved potting soil-sand mixture (Fig. 1). However, such dif-
ferences were not detected when plants were grown on clay
soil or when the soil was autoclaved before use. Abundance of
Pseudomonas spp. was not significantly different in the rhizos-
pheres of JA- or ET-signaling mutants (Fig. 2). Likewise,
rhizosphere population densities of total bacteria or
Pseudomonas spp. of ET-insensitive Tetr18 tobacco were not
different from those of wild-type tobacco (data not shown).
Soil type is one of the major factors determining bacterial
community structure and activity in the rhizosphere (Berg and
Smalla 2009; Garbeva et al. 2004a). Since differences in the
bacterial abundances on the jar1 and ein2 roots were not de-
tectable in the clay soils or when a new microflora was estab-
lished after autoclaving, the decreased abundance is probably
the result of the combination of plant genotype and other fac-
tors, such as extant microbial composition or edaphic variables.
Indeed the eubacterial community structure of the nonauto-
claved potting soil-sand mixture differs from that of the other
soil types (Doornbos 2009). Marschner and associates (2001)
experienced comparable bacterial community stability in a clay
soil. Whereas their study found that root zone was an important
factor for the composition of the rhizosphere communities in a
sandy and a sandy loam soil, this was of little importance for
the rhizosphere composition in the clay soil.
Since few differences were apparent in the absence of patho-
gens when defense signaling pathways are not activated, dif-
ferences might only be expressed when plant defenses have
been activated. Metabolic profiling of Arabidopsis roots upon
chemical activation of plant defenses displayed secretion of
numerous secondary metabolites, of which several have anti-
bacterial and antifungal activity at the concentrations detected
Fig. 6. Ordination biplots generated by principal component analysis of Pseudomonas spp.–specific denaturing gradient–gel electrophoresis fingerprints of
the rhizosphere of tobacco wild type and its ethylene-insensitive transformant Tetr18. A, Plants grown on potting soil and sand mixture. B, Plants grown on
autoclaved potting soil and sand mixture. C, Plants grown on clay soil. D, Plants grown on autoclaved clay soil. Crosses represent individual samples,
triangles the centroid position of the genotypes.
402 / Molecular Plant-Microbe Interactions
in the exudates (Walker et al. 2003b). For example, o-coumaric
acid was secreted after JA treatment and has in vitro antimicro-
bial activity against Rhizoctonia solani, Erwinia carotovora,
and Erwinia amylovora.
Activation of the JA-dependent signaling pathway by foliar
application of MeJA resulted in local and systemic expression
of the JA-responsive gene VSP2 (Fig. 7). However, VSP2 ex-
pression in roots was stimulated not only by JA but also, to a
lesser extent, by SA and its functional analog BTH. In spite of
the activation of the JA-dependent defense-signaling pathway,
no significant changes in the abundance of total culturable bac-
teria or Pseudomonas spp. were apparent in the rhizospheres
of Col-0, jar1, npr1, or NahG (Fig. 8). Thus, the induction of
ISR does not influence the density of the Arabidopsis
In all soils used, we observed a consistently lower number
of total bacteria and Pseudomonas spp. on the roots of the
cpr1 mutant (Fig. 1 and 2). The cpr1 mutant contains a high
level of SA, resulting in constitutive expression of SA-depend-
ent defenses (Bowling et al. 1994). As a result, the cpr1 mutant
has increased resistance to a variety of pathogens, including
the oomycete Hyaloperonospora arabidopsidis and the bacte-
rium P. syringae pv. maculicola (Bowling et al. 1994). This
expression of defenses has energy costs involved (Heidel et al.
2004; Van Hulten et al. 2006; Walters and Heil 2007), resulting
in a dwarf phenotype of the cpr1 mutant. To dissect the effect
of activated SA signaling from the effect on plant growth, SA-
dependent defenses were activated in wild-type plants by foliar
application of SA or BTH, as reflected by local and systemic
expression of the SAR marker gene PR-1 (Fig. 7). Under these
conditions, the activated SA-dependent defenses did not result
in changes in the abundance of total culturable bacteria or
Pseudomonas spp. in the rhizospheres of Col-0, npr1, or jar1
(Fig. 8A through C). However, numbers of Pseudomonas spp.
were increased in the rhizosphere of NahG (Fig. 8D). Because
the NahG gene encodes salicylate hydroxylase, these plants
convert SA to catechol (Delaney et al. 1994; Van Wees and
Glazebrook 2003). In bacteria, NahG is part of the operon
responsible for degradation of naphthalene, by which many
pseudomonads can use aromatics as a carbon source (Yen and
Serdar 1988; Zhao et al. 2005). It is possible and perhaps even
likely that increased population densities of Pseudomonas spp.
in the rhizosphere of SA-treated NahG plants result from in-
creased levels of catechol in the rhizosphere. Also, BTH treat-
ment may have increased SA content and, consequently, cate-
chol levels in NahG plants. In strawberry, BTH treatment has
been reported to increase SA content (Hukkanen et al. 2007);
however, this does not occur in tobacco (Friedrich et al. 1996).
In Arabidopsis, levels of phenolic metabolites are significantly
reduced in BTH-treated plants (Hien Dao et al. 2009), but spe-
cific data for SA are lacking.
Effect of plant defense signaling
on the bacterial community structure.
Whereas effects of activated defense signaling on bacterial
abundances were very small, these might still have affected bac-
terial diversity. Analysis of community structure of total bacteria
and Pseudomonas spp. by PCR-DGGE and multivariate analysis
demonstrated consistent clustering of the JA and ET mutants
jar1, etr1, and ein2 opposite to the Col-0 wild type (Figs. 3 and
4). In nonautoclaved potting soil and sand mixture the commu-
nity structures of total bacteria and Pseudomonas spp. in the
rhizospheres of NahG and jar1 differed significantly from those
of Col-0, as determined by ANOSIM analysis of the DGGE fin-
gerprints (Fig. 9; Table 3). Thus plant genotype does affect bac-
terial communities, but the effects are not consistent and there is
no apparent relation to plant defense signaling.
In ET-insensitive Tetr18 tobacco, Geraats (2003) observed
that differences in the community structure of root-associated
bacterial populations between wild type and the transformant
were already detectable before spontaneous development of
disease symptoms occurred. These symptoms occurred only
when plants were grown in nonautoclaved soil; autoclaving the
soil effectively reduced the population of deleterious oomy-
cetes (Pythium spp.) held responsible for the wilting and root
rot observed (Geraats et al. 2003; Knoester et al. 1998). In line
with those results, we observed a more distinct difference of
both eubacterial- and Pseudomonas spp. rhizosphere commu-
nities between Tetr18 and wild-type tobacco on nonautoclaved
soils than on the soils that were autoclaved before use (Figs. 5
and 6). Although disease symptoms were not observed in our
experiments, the shifts in the bacterial community structure
may have been due to early stages of root infection causing
changes in root exudates of the Tetr18 plants.
Activation of SA-dependent defenses did not alter total bac-
terial or Pseudomonas spp. community structure in the rhizos-
phere of Arabidopsis wild-type plants (Fig. 9). Whereas we
observed reduced bacterial abundance in the rhizosphere of the
cpr1 mutant, a reduction of the bacterial diversity in the cpr1
rhizosphere was not apparent. This observation is in line with
observations made by Hein and associates (2008), who could
not demonstrate a postulated decrease of bacterial diversity in
the cpr1 rhizosphere by terminal restriction fragment length
polymorphism. The cpr1 mutant has constitutively activated
SA-dependent defense responses. Thus, it must be concluded
that SA-activated SAR had no significant effect on bacterial
and Pseudomonas rhizosphere community structures in the
rhizosphere of Arabidopsis.
Fig. 7. Expression levels of A, the jasmonate-responsive gene VSP2 and B,
the salicylic acid–responsive gene PR-1 in Arabidopsis Col-0 leaves (local
effect) and roots (systemic effect) 24 h after foliar application of 0.1 mM
methyl jasmonate, 1 mM salicylic acid, or benzothiadiazole (200 µg per
milliliter) compared with mock-treated control (set at 1). Plants grown on
nontreated potting soil-sand mixture. Cycle threshold change compared
with mock-treated controls was as follows. VSP2leaves: 1.83, VSP2roots:
4.45, PR-1leaves: 0.88, and PR-1roots: 6.6.
Vol. 24, No. 4, 2011 / 403
Taken together, mutant and transgenic derivatives of Arabi-
dopsis affected in ISR or SAR expression did have altered bac-
terial or Pseudomonas spp. population densities in their rhizos-
phere, but no consistent impact on community structure of
rhizosphere bacteria was apparent. Moreover, activating the JA
or SA defense–signaling pathway by exogenous application of
MeJA, SA, or BTH had no effect on the bacterial rhizosphere
microflora, either. In tobacco, differences in the bacterial com-
munity structure were observed between wild type and ET-
insensitive Tetr18 plants, but only when these plans were grown
Fig. 8. Numbers (log CFU per gram) of total culturable bacteria and Pseudomonas spp. in the rhizosphere of Arabidopsis Col-0 and its derivatives affected in
defense signaling 2 weeks after foliar treatment with 0.1 mM methyl-jasmonate, 1 mM salicylic acid, or benzothiadiazole (200 µg per milliliter). Plants were
grown on a potting soil-sand mixture. Rhizosphere samples of five individual plants were dilution plated on 1/10 TSA+ (3 g of tryptic soy broth per liter, 13 g
of agar technical per liter, and 100 mg of natamycin per liter) and KB+ (King’s B agar supplemented with 13 µg of chloramphenicol per milliliter, 40 µg of
ampicillin per milliliter, and 100 µg of natamycin per milliliter). Different letters indicate significant differences (analysis of variance and Tukey post-hoc
test; P < 0.05), capitals and lower-case letters indicate a separate comparison; error bars represent standard errors.
Fig. 9. Ordination biplots generated by redundancy analysis of A, eubacterial- and B, Pseudomonas spp.–specific fingerprints of the rhizosphere of the
Arabidopsis genotypes Col-0 and its derivatives affected in defense signaling 2 weeks after foliar treatment with 0.1 mM methyl-jasmonate (crosses), 1 mM
salicylic acid (small triangles), or benzothiadiazole (200 µg per milliliter) (rectangles), or mock treated (squares). Large triangles represent the centroid posi-
tion of genotypes per treatment.
404 / Molecular Plant-Microbe Interactions
on soils likely to contain opportunistic pathogens. Therefore, the
observed differences in tobacco seem to be due to the increased
sensitivity of the ET-insensitive Tetr18 plants to infection rather
that to defective ET signaling per se. In contrast, differences
observed in the rhizospheres of the different Arabidopsis
genotypes do not appear to be related to plant defense. There-
fore, control of plant diseases by activation of ISR or SAR is
unlikely to have a significant impact on the indigenous soil
MATERIALS AND METHODS
Cultivation of plants.
Seeds of Arabidopsis thaliana wild type, accession Col-0, and
the JA, ET, or SA signal-transduction mutants jar1-1, etr1-1,
ein2-1, cpr1-1, npr1-1, and NahG (Table 4) as well as seeds
from Nicotiana tabacum cv. Samsun NN and its ET-insensitive
transformant Tetr18 were sown in autoclaved (20 min at 121°C)
sand. Seeds were kept at 4°C in the dark for 2 days and were
subsequently allowed to germinate in a greenhouse with a cycle
of an 8-h day (200 μE m–2 s–1) at 24°C and 16-h night at 20°C
and 70% relative humidity. After two weeks, germinated seed-
lings were transferred individually to 60-ml pots containing
either a potting soil-sand mixture (12:5 vol/vol) (Pieterse et al.
1996) or a clay soil (Glandorf et al. 2001; Viebahn et al. 2003).
Both soils were either nonautoclaved or autoclaved (twice for 20
min at 121°C with a 24-h interval). The transferred seedlings
were grown under the conditions described above. Plants were
watered with half-strength Hoagland nutrient solution
(Hoagland and Arnon 1938) once a week and with tap water as
required. All samples were harvested from 5-week-old plants.
No differences were noticed between plants grown on auto-
claved or nonautoclaved soils, but plants grew more slowly on
the clay than on the potting soil-sand mixture.
Foliar application of MeJA, SA, and BTH.
Application of MeJA induces JA-dependent ISR (Pieterse et
al. 1998), while application of SA or its functional analog
BTH results in SAR (Gaffney et al. 1993; Lawton et al. 1996).
One day before treatment, plants were put at 100% relative
humidity. The leaves of 3-week-old plants were briefly sub-
merged in an aqueous solution of either 0.1 mM MeJA, 1 mM
SA, or BTH (200 µg per milliliter), were supplemented with
0.015% Silwet L-77 (Van Meeuwen Chemicals BV, Weesp,
The Netherlands), avoiding spillover to the soil. Leaves of con-
trol plants were briefly submerged in 0.015% Silwet L-77. Af-
ter treatment, plants were further grown in the greenhouse as
described above. After 24 h, leaves and roots were harvested
from some of the plants and were frozen in liquid nitrogen and
stored at –80°C for analysis of marker gene expression. Two
weeks after chemical treatment, rhizosphere samples were har-
vested from the remaining plants for bacterial quantification
and PCR-DGGE analysis.
Quantification of bacteria.
For five individual plants per treatment, roots with tightly
adhering soil were harvested and were shaken three times in
10 mM MgSO4 with glass beads (0.6 to 0.8 mm) on a vortex at
maximum speed for 1 min. Population densities of aerobic
heterotrophic bacteria were determined by plating serial dilu-
tions on 1/10 strength TSA+ (3 g of tryptic soy broth per liter
[Difco Laboratories, Detroit], 13 g of agar technical per liter
[Oxoid Ltd, Basingstoke, U.K.] and 100 mg of natamycin per
liter [Pedersen 1992] [Delvocid; DSM, Delft, The Nether-
lands] and counting CFU after 7 days of incubation at 20°C.
Quantification of Pseudomonas spp. was performed by plating
appropriate dilutions on KB+ (King’s B agar [King et al. 1954]
supplemented with 13 µg of chloramphenicol per milliliter, 40
µg of ampicillin per milliliter, and 100 µg of natamycin per
milliliter [Delvocid; DSM] [Geels and Schippers 1983]) and
counting CFU after incubation for 48 h at 28°C.
DNA extraction and PCR-analysis.
Two replicate samples from five pooled individual plants each
were analyzed for every treatment. DNA was extracted from the
rhizosphere as described by Viebahn and associates (2005a).
Briefly, 500 mg of root material with adhering soil was used for
direct DNA extraction with the FastDNA SPIN kit for soil
(Bio101; Biogene, Vista, CA, U.S.A.), according to the manu-
facturer’s instructions. Bead beating was performed with a Ribo-
lyser (Hybaid, Ashford, U.K.), and for the final elution, 100 μl
of sterile water was used instead of 50 μl. DNA extracts were
purified with the Wizard DNA clean-up system (Promega, Madi-
son, WI, U.S.A.) according to the manufacturer’s protocol. Am-
plification of the V6-V8 region of eubacterial 16S rDNA genes
was done with primers 968f_GC and 1401R (Nübel et al. 1996).
The PCR reaction mix consisted of 1× PCR buffer 2 (Roche
Diagnostics, Mannheim, Germany), 250 μM of each dNTP, 200
nM of each primer, 2.5 U Expand Long Template enzyme
(Roche Diagnostics), and 1 μl of purified DNA extract. PCR
conditions used in the thermocycler (Hybaid, Ashford, U.K.)
were 5 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1
min at 66°C, and 3 min at 72°C, and a final extension of 10 min
at 72°C. PCR specific for Pseudomonas spp. was performed
Table 4. Listing and relevant characteristics of Arabidopsis lines used
Genotype Relevant characteristicsa Reference
Wild-type accession Colombia-0
jasmonate resistance 1, JA-insensitive
ethylene response 1, ET-insensitive
ethylene insensitive 2, ET-insensitive
nonexpressor of PR genes, impaired in SA- and JA/ET-dependent defense responses
constitutive expressor of PR genes 1, SA overproducer
Transformant expressing bacterial SA hydroxylase, does not accumulate SA
Staswick et al. 1992
Bleecker et al. 1988
Guzman and Ecker 1990
Cao et al. 1994; Pieterse and Van Loon 2004
Bowling et al. 1994
Delaney et al. 1994
a JA = jasmonate, ET = ethylene, PR = pathogenesis related, and SA = salicylic acid.
Table 3. Two-way analysis of similarity of denaturing gradient–gel electrophoresis fingerprints of eubacteria and Pseudomonas spp. in the rhizospheres of
Arabidopsis genotypes (Col-0, NahG, jar1, and npr1) that were treated with different hormones (salicylic acid, benzothiadiazole, methyl jasmonate)
Values Genotype Treatment Genotype Treatment
Global R value
Vol. 24, No. 4, 2011 / 405
with 16S rDNA primers PsF and PsR (Widmer et al. 1998). To
obtain Pseudomonas-specific amplicons suitable for separation
by DGGE, nested PCR on 50-fold diluted primary PCR product
was performed according to Garbeva and associates (2004b). All
amplicons were checked for size and concentration by electro-
phoresis on a 1.0% agarose gel in 1× TAE (40 mM Tris-acetate,
20 mM Na-acetate, and 1.0 mM Na-EDTA, pH 8.0) (Sambrook
et al. 1989).
PCR amplicons were separated on polyacrylamide gels with
a 30 to 60% denaturant gradient (8% acrylamide, acrylamide/
bisacrylamide ratio 37.5:1; 100% denaturing solution is 7 M
urea, 40% formamide). Depending on amplicon concentration,
15 to 25 μl of PCR products were loaded in random order.
Gels were run in 0.5× TAE at 60°C for 17 h at 80 V in a
DCode universal mutation detection system (Bio-Rad Labora-
tories, Veenendaal, The Netherlands). Gels were stained with
1:10,000 diluted CyberGold (Molecular Probes, Leiden, The
Netherlands) in 0.5× TAE and were photographed on a blue-
light transilluminator (Clare Chemical Research, Dolores, CO,
U.S.A.) with a GeneGenius Bioimaging System (Syngene,
RNA extraction and quantitative PCR analysis.
RNA extraction and quantitative PCR analysis were per-
formed according to Van der Ent and associates (2008) and
Czechowski and associates (2004). Briefly, frozen plant material
of two replicates of two plants each was pulverized in a mortar
with pestle. From a 500-µl aliquot, total RNA was extracted by
the addition of an equal volume of extraction buffer (0.35 M
glycine, 0.0048 M NaOH, 0.34 M NaCl, 0.04 M EDTA, 4%
(wt/vol) sodium dodecyl sulfate). The homogenates were
extracted twice with an equal volume of phenol/chloroform/iso-
amylalcohol (25:24:1), followed by an extraction with an equal
volume of chloroform/isoamylalcohol (24:1). RNA was precipi-
tated with LiCl and was purified by NaAc (pH 5.2) and ethanol
precipitation, according to Sambrook and associates (1989).
RNA (2 µg) was DNAse treated (Ambion, Huntingdon, U.K.)
according to the manufacturer’s instructions. To check for con-
tamination with genomic DNA, a PCR with primers for EIL2
(At5g21120) was carried out (EIL2fw: TCT CGT GAG ACG
GTC TAG AAG TT; EIL2rev: ATG AAA CCT AAT CTT CTC
CAT TGC), as described by Van der Ent and associates (2008).
DNA-free total RNA was converted to cDNA, using oligo
dT20 primers (Invitrogen, Breda, The Netherlands), dNTPs,
and SuperScript III reverse transcriptase (Invitrogen, Breda,
The Netherlands), according to the manufacturer’s instruc-
tions. Quantitative PCR reactions took place in optical 96-well
plates with a MqIQ Single Color real-time PCR detection sys-
tem (Bio-Rad), using Power SYBR Green PCR master mix
(Applied Biosystems, Warrington, U.K.) according to the
manufacturer’s instructions, with primers designed for PR-1
(At2g14610: PR-1fw, CTC GGA GCT ACG CAG AAC AA;
PR-1rev, TTC TCG CTA ACC CAC ATG TTC A) and VSP2
(At5g24770: VSP2fw, TCG AAG TTG ACG CAA GTG GT;
VSP2rev, GGG GAC AAT GCC ATG AAG AT). Expression of
PR-1 and VSP2 was corrected for the constitutively expressed
reference gene At1g13320 encoding protein phosphatase 2A
subunit A3 (At1g13320fw: TAA CGT GGC CAA AAT GAT
GC; At1g13320rev: GTT CTC CAC AAC CGC TTG GT), after
which expression levels were calculated with respect to the
Bacterial plate counts were 10log transformed and were ana-
lyzed by analysis of variance and Tukey post-hoc test (P <
0.05) (SPSS v16.0). Power analysis was performed with PASS
2008 (NCSS, Kaysville, Utah, U.S.A.). To study effects of
plant genotype and hormone treatments on the microbial com-
munity, we used redundancy analysis (Ramette 2007). The
analysis was performed with Bionumerics v4.5 program (Ap-
plied Maths, Sint-Martens,-Latem, Belgium) and Canoco v4.5
(Microcomputer Power, Ithaca, NY, U.S.A.), as described pre-
viously (Costa et al. 2006). Briefly, images were aligned and
standardized with Bionumerics v4.5 prior to generation of the
presence or absence and relative peak intensities data of differ-
ent band positions within each lane. Subsequently, Canoco
v4.5 was used for ordination of DGGE fingerprints and envi-
ronmental variables in the biplots. Detrending corresponding
canonical analysis (DCCA) was applied to check for heteroge-
neity within the data set. Since DCCA analysis indicated a
homogeneous data set, the use of RDA or principal component
analysis was used as the most appropriate algorithm to generate
the ordination plots. In the ordination plots, distances between
symbols represent a measure of similarity in that symbols that
lie close together have a similar community structure, whereas
a large distance between symbols indicates differences in com-
munity structure. The x axis corresponds to the first eigenvalue
and explains a higher percentage of the total variance than the
y axis, which corresponds to the second component. Thus, the
first component is more important for explaining the variance
than the second (Marschner and Rumberger 2004). In case of
RDA analysis, Monte Carlo permutation tests were performed
to assess the significance of DGGE-derived fingerprints and
environmental correlations, assuming that DGGE ribotype is
not related to environment. One- or two-way analysis of simi-
larity (ANOSIM) was subsequently performed to compare
microbial community structures as affected by plant genotype,
hormone treatments, or both. The distance measure used was
Bray-Curtis. The significance was examined by a permutation
test where R was recomputed for 10,000 permutations. Results
from pairwise ANOSIM were analyzed to interpret the main
between-group differences: R > 0.75 separated bacterial and
Pseudomonas compositions, R > 0.5 was considered as over-
lapping but clearly different, and R < 0.25 as barely separating.
Calculation of similarity coefficients and ANOSIM analysis
were carried out using PAST (Hammer et al 2001).
All experiments were performed at least twice with com-
We thank K. Wernars and B. van Rotterdam (the National Institute of
Public Health and the Environment, Bilthoven, The Netherlands) for their
hospitality to perform DGGE analysis and I. van der Tweel (Utrecht Uni-
versity) for performing the Power analysis. T. Van and J. Vervoort are
acknowledged for technical assistance.
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