Download Pseudomonas spp.-induced systemic resistance

Journal of Experimental Botany, Vol. 61, No. 1, pp. 249–260, 2010
doi:10.1093/jxb/erp295 Advance Access publication 7 October, 2009
RESEARCH PAPER
Pseudomonas spp.-induced systemic resistance to Botrytis
cinerea is associated with induction and priming of defence
responses in grapevine
Bas W. M. Verhagen1,*, Patricia Trotel-Aziz1, Michel Couderchet1, Monica Höfte2 and Aziz Aziz1,†
1
2
URVVC – Stress & Environment EA 2069, PPDD, University of Reims, F-51687 Reims cedex 2, France
Laboratory of Phytopathology, Faculty of Bioscience Engineering, University of Ghent, B-9000 Gent, Belgium
Received 1 July 2009; Revised 8 September 2009; Accepted 10 September 2009
Abstract
Non-pathogenic rhizobacteria Pseudomonas spp. can reduce disease in plant tissues through induction of a defence
state known as induced systemic resistance (ISR). This resistance is based on multiple bacterial determinants, but
nothing is known about the mechanisms underlying rhizobacteria-induced resistance in grapevine. In this study, the
ability of Pseudomonas fluorescens CHA0 and Pseudomonas aeruginosa 7NSK2 to induce resistance in grapevine
against Botrytis cinerea is demonstrated. Both strains also triggered an oxidative burst and phytoalexin
(i.e. resveratrol and viniferin) accumulation in grape cells and primed leaves for accelerated phytoalexin production
upon challenge with B. cinerea. Treatment of cell cultures with crude cell extracts of bacteria strongly enhanced
oxidative burst, but resulted in comparable amounts of phytoalexins and resistance to B. cinerea to those induced
by living bacteria. This suggests the production of bacterial compounds serving as inducers of disease resistance.
Using other strains with different characteristics, it is shown that P. fluorescens WCS417 (Pch-deficient), P. putida
WCS358 (Pch- and SA-deficient) and P. fluorescens Q2-87 (a DAPG producer) were all capable of inducing resistance
to an extent similar to that induced by CHA0. However, in response to WCS417 (Pch-negative) the amount of H2O2
induced is less than for the CHA0. WCS417 induced low phytoalexin levels in cells and lost the capacity to prime for
phytoalexins in the leaves. This suggests that, depending on the strain, SA, pyochelin, and DAPG are potentially
effective in inducing or priming defence responses. The 7NSK2 mutants, KMPCH (Pch- and Pvd-negative) and
KMPCH-567 (Pch-, Pvd-, and SA-negative) induced only partial resistance to B. cinerea. However, the amount of
H2O2 triggered by KMPCH and KMPCH-567 was similar to that induced by 7NSK2. Both mutants also led to a low
level of phytoalexins in grapevine cells, while KMPCH slightly primed grapevine leaves for enhanced phytoalexins.
This highlights the importance of SA, pyochelin, and/or pyoverdin in priming phytoalexin responses and induced
grapevine resistance by 7NSK2 against B. cinerea.
Key words: Induced resistance, oxidative burst, phytoalexins, priming, Pseudomonas spp., rhizobacteria, siderophores,
Vitis vinifera.
Introduction
Induced resistance is a state of enhanced defensive capacity
of plants in order to mobilize appropriate cellular defence
responses before or upon pathogen attack (Bakker et al.,
2007). It is generally systemic and can be triggered by
microbial-associated molecular patterns (MAMPs) or
several non-pathogenic rhizobacteria such as Pseudomonas
spp. The molecular mechanisms involved in rhizobacteriainduced systemic resistance (ISR) appear to vary among
* Present address: MAF Biosecurity New Zealand, PO Box 2526, Wellington 6140, New Zealand.
y
To whom correspondence should be addressed: E-mail: [email protected]
Abbreviations: AOS, active oxygen species; DAPG, 2;4-diacetylphloroglucinol; ISR, induced systemic resistance; LPS, lipopolysaccharides; MAMPs, microbeassociated molecular patterns; Pch, pyochelin; Pvd, pyoverdin; SA, salicylic acid.
ª The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
250 | Verhagen et al.
bacterial strains and pathosystems. Ton et al. (1999) showed
that it is possible to trigger ISR with Pseudomonas
fluorescens WCS417 in eight Arabidopsis accessions, but not
in the accessions RLD and Ws-0. Furthermore, the ability
of bacteria to enhance resistance appears to be plant–
bacteria specific, since P. putida WCS358 induced resistance
in Arabidopsis, but not in radish (Van Peer et al., 1991;
Leeman et al., 1995; Van Wees et al., 1997). Finally the
induced resistance appears to be effective against several,
but not all pathogens, depending on the resistance that is
effective against these pathogens (Pieterse et al., 1996; Van
Wees et al., 1997; Ton et al., 2002). Treatment of roots with
the ISR-triggering bacteria P. fluorescens WCS417 leads to
many local changes in gene expression, but not all are
involved in ISR (Verhagen et al., 2004).
In plants exhibiting ISR, a number of reactions have been
observed such as deposition of callose, lignin, and phenolics
beyond infection sites (Duijff et al., 1997), increased
activities of chitinase, peroxidase, polyphenol oxidase, and
phenylalanine ammonia lyase (Chen et al., 2000; MagninRobert et al., 2007), enhanced phytoalexin production (Van
Peer et al., 1991), and induced expression of stress-related
genes (Verhagen et al., 2004). The enhanced defensive
capacity of induced plants does not necessarily require de
novo defences but can also result from a faster and/or
stronger expression of basal defence responses upon pathogen attack. This enhanced capacity to express infectioninduced basal defences is called priming (Conrath et al.,
2002), the fitness costs of which are substantially lower than
those of constitutively activated defences (Van Hulten et al.,
2006).
Successful establishment of ISR depends on the recognition of bacterial determinants by the plant roots. Several
bacterial traits operative in triggering ISR have been
identified. These included flagellin, lipopolysaccharides
(LPS), antibiotics, quorum-sensing molecules, cyclic
lipopeptides, volatiles, and siderophores (Bakker et al.,
2007; Ongena et al., 2007; Tran et al., 2007). The
involvement of these compounds in ISR can differ between
plants and bacteria. In Arabidopsis, LPS, siderophores, and
flagellin of P. putida WCS358 are involved in the induction
of resistance, while in bean and tomato only the LPS and
siderophores appeared to be active (Meziane et al., 2005). In
Arabidopsis both P. fluorescens CHA0 and P. fluorescens
Q2-87 use 2,4-diacetylphloroglucinol (DAPG) in the
induction of resistance (Iavicoli et al., 2003). P. fluorescens
WCS417 uses an iron-regulated compound and the LPS to
induce resistance in radish (Leeman et al., 1995), but the
only LPS is important in Arabidopsis and carnation (Van
Wees et al., 1997). Pseudomonas aeruginosa 7NSK2 has also
been demonstrated to induce ISR in several plant species,
including bean, tobacco, tomato, and rice (De Meyer and
Höfte, 1997; De Meyer et al., 1999; Audenaert et al., 2002;
De Vleesschauwer et al., 2006). This resistance has been
linked to the production of SA (De Meyer et al., 1999), the
SA-derived pyochelin and/or the phenazine pyocyanin by
7NSK2 (Audenaert et al., 2002; De Vleesschauwer et al.,
2006).
In grapevine, reports about the induction of systemic
resistance using beneficial micro-organisms are scarce
(Compant et al., 2005; Magnin-Robert et al., 2007; TrotelAziz et al., 2008). Nevertheless, grapevine plants can express
various defence mechanisms during interaction with pathogenic micro-organisms (Busam et al., 1997; Bézier et al.,
2002; Robert et al., 2002) and in response to elicitor
molecules that enhance resistance against Botrytis cinerea
and Plasmopara viticola (Aziz et al., 2006, 2007; Vandelle
et al., 2006; Varnier et al., 2009). These defence responses
comprise the oxidative burst characterized by the transient
generation of active oxygen species (AOS), the accumulation of host-synthesized phytoalexins and the accumulation
or increased activity of pathogenesis-related (PR) proteins
with hydrolytic activity (e.g. chitinases and glucanases).
Recently, it has been suggested that generation of AOS is
one of the earliest events produced in cell suspension
cultures in response to rhizobacterial elicitors that could be
linked to the development of ISR in whole plants (Van
Loon et al., 2008). This oxidative burst was similar to that
induced by oligosaccharides in grapevine suspension cells
(Aziz et al., 2004, 2007), in which this response was found
to be associated with increased defence-related gene expression and induced resistance in intact plants (Aziz et al.,
2003). Hydrogen peroxide had a direct toxic effect on
micro-organisms, and is also needed for the strengthening
of the cell walls and cell wall protein cross-linking (Lamb
and Dixon, 1997), or can diffuse across cell membranes and
has therefore been implicated in the signalling for the
establishment of downstream plant immunity events
(Levine et al., 1994).
Stilbenic phytoalexins have attracted considerable attention in grapevine because they can be considered as markers
for plant disease resistance and have been shown to possess
biological activity against a wide range of pathogens
(Coutos-Thevenot et al., 2001; Jeandet et al., 2002; Delaunois et al., 2009). The most studied are trans-resveratrol
(3,5,4#-tryhydroxystilbene), its oligomers viniferins, and its
derivatives known as piceide (5,4#-dihydroxystilbene-3-Ob-glucopyranoside) and pterostilbene (3,5-dimethoxy-4#hydroxystilbene) (Jeandet et al., 2002; Pezet et al., 2004).
These stilbenic compounds are selectively accumulated in
leaves and grape skins in response to fungal infections, UV
radiation, elicitors or chemicals (Jeandet et al., 2002; Aziz
et al., 2003, 2006).
In this study, the well-known ISR triggering bacteria,
Pseudomonas fluorescens CHA0 and P. aeruginosa 7NSK2
were investigated for their ability to enhance grapevine
resistance to subsequent infection with B. cinerea. To assess
bacterial determinants in this process, the effect of
P. fluorescens CHA0 was compared with those of other
strains such as P. fluorescens WCS417 (Pch-deficient),
P. fluorescens Q2-87 (DAPG producer), and P. putida
WCS358 (Pch- and SA-deficient) (Table 1). Similarly, the
inducing activity of P. aeruginosa 7NSK2 was compared
with that of its derivative mutants KMPCH (Pch- and Pvddeficient) and KMPCH-567 (Pch-, Pvd-, and SA-deficient).
The effects of crude bacterial extracts or growing medium
Pseudomonas-mediated ISR in grapevine | 251
Table 1. Bacterial strains and mutants used in this study with their relevant characteristics
Pseudomonas sp. strain
Origin and relevant characteristicsa
Reference or source
Pseudomonas fluorescens CHA0
Isolated from tobacco rhizosphere; DAPG+, Plt+,
Prn+, HCN+, AprA+
Isolated from wheat rhizosphere; OA+,
Pvd+, Pch-, SA+
Isolated from wheat roots; DAPG+
Isolated from potato rhizosphere; OA+, Pvd+,
Pch-, SAIsolated from bean rhizosphere; Pvd+, Pch+,
SA+, Pyo+,
Pyo+, Pvd–, Pch–, SA+, chemical mutant of the
pyoverdine-negative mutant MPFM1, Kmr
Pvd–, Pch–, SA–, pchA replacement mutant of
KMPCH; Kmr
Voisard et al., 1994; Iavicoli et al., 2003
Pseudomonas fluorescens WCS417
Pseudomonas fluorescens Q2-87
Pseudomonas putida WCS358
Pseudomonas aeruginosa 7NSK2
Pseudomonas aeruginosa KMPCH
Pseudomonas aeruginosa KMPCH-567
Lamers et al., 1988; Leeman et al., 1995
Raaijmakers et al., 1997
Geels and Schippers, 1983; Van Vees et al., 1997
Iswandi et al., 1987
Höfte et al., 1993
De Meyer et al., 1999
a
Pvd, pyoverdin; Pch, pyochelin; SA, salicylic acid; Pyo, pyocyanin; DAPG, 2;4-diacetylphloroglucinol; Plt, pyoluteorin; Prn, pyrrolnitrin; AprA,
exoprotease; OA, O-antigenic side chain of lipolysaccharide; Km, kanamycin.
were also evaluated and some defence responses, such as the
production of active oxygen species (AOS) and phytoalexin
accumulation, were examined in suspension-cultured cells of
grapevine. Phytoalexins were also examined in leaves of
intact plantlets before and upon B. cinerea challenge, and
related to the effectiveness of selected bacterial strains to
induce systemic resistance.
Materials and methods
Cultivation of rhizobacteria
Bacterial strains and mutants used in this study are listed in Table
1. Pseudomonas fluorescens CHA0, WCS417, Q2-87, P. putida
WCS358, P. aeruginosa 7NSK2 and its mutants KMPCH and
KMPCH-567 bacteria were grown in liquid Luria-Bertani (LB) or
King B (KB) medium (King et al., 1954), for 24 h at 28 C and 120
rpm as described previously (Pieterse et al., 1996). Bacteria were
collected by centrifugation and resuspended in 10 mM MgSO4 at
105, 106, and 107 CFU ml1. To check the effects of the medium in
which bacteria have grown, the medium fraction was collected as
a supernatant after centrifugation of each suspension of live
bacteria. The remaining pellet containing bacteria were resuspended in MgSO4 and boiled at 95 C for 15 min to obtained
crude cell extracts from different initial concentrations of live
bacteria. Both bacterial extracts and media were checked for the
absence of bacterial cells by plating them onto LB or KB agar
plates.
Fungal pathogen
Botrytis cinerea strain 630 was cultured in Erlenmeyer flasks
containing potato dextrose agar (Sigma) for 4 weeks at 22 C.
Spores were collected and resuspended in sterile distilled water to
a final density of 106 conidia ml1. The spores were used
immediately for the inoculation of leaves as described previously
(Aziz et al., 2003) or stored at –80 C until use.
Plant growth conditions and cell cultures
Grapevine plantlets (Vitis vinifera cv. Chardonnay 7535) were
obtained in vitro by multiplication through in vitro micro-cuttings
on modified Murashige and Skoog (1962) medium, as described
previously (Aziz et al., 2003). Plantlets were grown at 24 C with
16/8 h light/dark at 70% humidity. Grapevine cells (Vitis vinifera
cv. Gamay) were cultivated in the medium of Nitsch and Nitsch
(1969) without hormones on a rotary shaker (130 rpm) at 25 C in
continuous light. Cells were maintained in the exponential phase
and subcultured 24 h prior to use.
Protection assays on detached leaves
Leaves were excised from 10-week-old in vitro-grown grapevine
plantlets and treated by floating them, adaxial side downward, on
standard buffer (2 mM MES, pH 5.9, containing 0.5 mM CaCl2
and 0.5 mM K2SO4), containing live bacteria or their corresponding extracts or media, with equivalent initial concentrations of live
bacteria. After 48 h, leaves were rinsed with distilled water and
placed on wet absorbing paper in glass Petri dishes. One needleprick wound was applied to the abaxial side of each leaf and
covered with 7.5 ll drops of a conidial suspension of B. cinerea
(106 conidia ml1). Quantification of disease development was
measured as the average diameter of lesions formed at 5 d postinoculation, and the average disease in the control was used to
calculate the % of disease reduction.
Induced systemic resistance
Four-week-old grapevine plantlets were uprooted and treated by
dipping the root system in a 20 ml suspension of live bacteria (107
CFU ml1) or corresponding cell extracts in 10 mM MgSO4 for
15 s before transplanting into a new medium of Murashige and
Skoog (1962). After 15 d or 28 d, the leaves were detached from
treated and control plantlets and challenge inoculated with
B. cinerea as described before. The average lesion size was
determined after 5 d, and the % disease reduction was calculated
by comparing the treatments with the average lesion size in the
challenged control.
Treatment of cell suspensions
Cells were collected during the exponential growth phase and
washed by filtration in a suspension buffer containing 175 mM
mannitol, 0.5 mM K2SO4, 0.5 mM CaCl2, and 2 mM MES, pH
5.5. Cells were resuspended at 0.1 g FW ml1 with suspension
buffer and equilibrated for 2 h on a rotary shaker (130 rpm,
24 C). Grapevine cells were then treated with live bacteria,
bacterial extracts or culture media at equivalent concentrations to
live bacteria. Control cells were incubated under the same
conditions and treated with MgSO4 solution. Grapevine cells were
then used for measurements of H2O2 production, phytoalexin
accumulation, and cell death during treatments.
252 | Verhagen et al.
H2O2 production
The production of H2O2 by the cells was assayed by chemiluminescence using luminol as described in Aziz et al. (2003). Cells were
treated and chemiluminescence was measured in a 10 s period with
a luminometer (Lumat LB 9507, Berthold).
Cell viability was assayed using the vital dye Neutral Red as
described in Aziz et al. (2003).
Phytoalexin quantification
The isolation and analysis of phytoalexins in cell cultures was
performed as described by Aziz et al. (2003). Stilbenes from the
cells (2 ml) were extracted in 2 ml of 85% methanol overnight at
4 C. After centrifugation (10 min, 5000 g), supernatants were
evaporated under nitrogen and the samples were solubilized in
1 ml of 100% methanol. Phytoalexins were analysed by HPLC
using a linear gradient of 10–85% acetonitrile at a flow rate of 1 ml
min1. Phytoalexins, trans-resveratrol and e-viniferin were
detected with a photodiode array detector coupled to a fluorometer
detector (kex ¼ 330 nm, kem ¼ 374 nm) and quantified on the
basis of standard calibration curves. The isolation and analysis of
phytoalexins from leaves follow the same procedures with slight
modifications as described by Aziz et al. (2006).
Statistical analysis
All experiments were done at least five times per treatment and
statistical analysis was performed by analysis of variance and
Duncan’s multiple range test, using SigmaStat 3.5 Systat Software.
Comparisons of statistical significance were made for each
sub-figure.
Results
Reduction of B. cinerea development in detached
leaves
The rhizobacteria P. fluorescens CHA0, P. fluorescens
WCS417, P. putida WCS358, and P. fluorescens Q2-87, as
well as P. aeruginosa 7NSK2 have been shown to induce
resistance in different plant species. To verify if these
rhizobacteria were capable of inducing resistance in grapevine, detached leaves were incubated for 48 h with the
different bacteria and afterwards washed and challenged
with the necrotrophic pathogen B. cinerea. Within 5 d after
inoculation, control leaves developed large necrotic lesions
(diameter >16 mm). By contrast, treatment of detached
leaves with most of the selected bacteria resulted in
a significant reduction of grey mould disease (Fig. 1A). This
reduction varied between 20–43% depending on the bacterium, WCS358 strain being the most active.
P. aeruginosa 7NSK2 reduced symptoms of B. cinerea by
30–40%. Likewise, the mutant KMPCH, defective in
pyochelin and pyoverdin, was equally as effective in
suppressing the disease as the parental strain. However, the
mutant KMPCH-567 lacking the pyoverdin, pyochelin, and
SA did not protect leaves against B. cinerea (Fig. 1B). The
inability of KMPCH-567 to reduce disease is not due to
insufficient colonization of the medium, because population
densities of 7NSK2 and its mutants are similar (1–1.23107
CFU ml1).
Crude cell extracts of bacteria were also used in
protection assays. As can be seen in Fig. 1C, D, most of
Fig. 1. Protection of grapevine leaves against Botrytis cinerea
after treatment with Pseudomonas spp. or with their crude cell
extracts. (A, B) Reduction of B. cinerea development in detached
leaves after treatment with living Pseudomonas fluorescens CHA0,
P. fluorescens WCS417, P. putida WCS358, and P. fluorescens
Q2-87 (A), and P. aeruginosa 7NSK2 and its mutants (B) at 107
CFU ml1, or in untreated leaves (Ctrl). (C, D) Reduction of B.
cinerea development in detached leaves after treatment with crude
extracts of P. fluorescens CHA0, P. fluorescens WCS417, P.
putida WCS358, and P. fluorescens Q2-87 (C), and P. aeruginosa
7NSK2 and its mutants (D) at an equivalent concentration of live
bacteria of 107 CFU ml1, or in untreated leaves (Ctrl). Data, as
a percentage of disease reduction compared to untreated leaves,
are the mean of at least five experiments, n >85. Comparisons of
statistical significance were made for each sub-figure. Different
letters indicate statistically significant differences between treatments (Duncan’s multiple range test; P <0.05).
the bacterial extracts were capable of reducing necrotic
lesions in leaves at a level comparable to that of live
bacteria. Although live cells of KMPCH-567 did not
provoke disease reduction (Fig. 1B), its killed cells induce
a slight, but significant protective effect against B. cinerea
(Fig. 1D). These results suggest that compounds produced
in vivo probably cannot be perceived by grape cells. Effects
of the bacterial extracts, which contain cell-wall fragments
also suggested a possible role of membrane LPS in reducing
disease.
Enhanced disease resistance in the leaves of treated
plants to B. cinerea
To rule out the possibility that the observed disease
protection was due to direct effects of bacteria on
B. cinerea, roots of in vitro-grown plantlets were dipped in
a suspension of live or killed bacteria, leaves were detached
2 weeks later and challenge-inoculated with B. cinerea.
Root-treatment with P. fluorescens CHA0, P. fluorescens
WCS417, P. putida WCS358, as well as P. fluorescens Q2-87
Pseudomonas-mediated ISR in grapevine | 253
led to an enhanced state of resistance against B. cinerea in
the leaves (Fig. 2A). Similarly, P. aeruginosa 7NSK2
significantly reduced disease development in the leaves
(Fig. 2B). The pyochelin negative mutant KMPCH (also
pyoverdin-deficient) reduced the disease to an extent slightly
higher than that induced by KMPCH-567, but reduction
remained smaller compared with their parental strain
7NSK2. Root treatments with bacterial extract preparations
were also effective as the live strains in inducing resistance
(Fig. 2C, D). This indicates that either live bacteria or
bacterial extracts are capable of inducing ISR in grapevine
against B. cinerea.
Induction of oxidative burst in grapevine cell
suspensions by rhizobacteria
To investigate if the oxidative burst might represent an
initial step in the elicitation of ISR (Van Loon et al., 2008),
the selected bacteria or their corresponding extracts (or
media) were tested for their effect on the production of
Fig. 2. Induced systemic resistance of grapevine plants against
Botrytis cinerea after root treatment with Pseudomonas spp. or
with their cell extracts. Plants were treated at the root level and
leaves were subsequently excised and inoculated with B. cinerea.
(A, B) Disease resistance in the leaves of grapevine plants treated
with living P. fluorescens CHA0, P. fluorescens WCS417, P. putida
WCS358, and P. fluorescens Q2-87 (A), and P. aeruginosa 7NSK2
and its mutants (B) at 107 CFU ml1, or in untreated plants (Ctrl).
(C, D) Disease resistance in the leaves of grapevine plants treated
with crude extracts of P. fluorescens CHA0, P. fluorescens
WCS417, P. putida WCS358, and P. fluorescens Q2-87 (C), and
P. aeruginosa 7NSK2 and its mutants (D) at an equivalent
concentration of live bacteria of 107 CFU ml1, or in untreated
plants (Ctrl). Data, as a percentage of disease reduction compared
to the leaves of untreated plants (Ctrl), are mean of at least five
experiments, n >70. Comparisons of statistical significance were
made for each sub-figure. Different letters indicate statistically
significant differences between treatments (Duncan’s multiple
range test; P <0.05).
H2O2 by grape cells. Addition of CHA0 and 7NSK2 to
grapevine cell suspensions triggered a single and transient
burst of active oxygen species (AOS) (Fig. 3A). The amount
of H2O2 increased steadily with the same time kinetic for
both strains until around 90 min, when a maximum was
reached at approximately 6 lM H2O2 in response to CHA0
and 3.5 lM H2O2 with 7NSK2. After this maximum, the
amount drops back until background levels. The dose–
response curves (Fig. 3B) show that no oxidative burst
occurred with 106 CFU ml1 corresponding to 105 CFU g1
grape cells, while a threshold for live cells of both strains at
107 CFU ml1 (equivalent to 106 CFU g1 grape cells) was
needed for the induction of a burst of H2O2.
The effect of CHA0 and 7NSK2 was also compared with
other bacterial strains that have different modes of inducing
resistance or that are affected in the expression of factors
known to be associated with ISR in other plant species
(Bakker et al., 2007). Results (Fig. 3C) show that
P. fluorescens WCS417, P. putida WCS358, and P. fluorescens Q2-87 were all capable of inducing an oxidative burst
with the same time kinetic than did CHA0 and 7NSK2, but
to different extents. A high level of H2O2 (50–55 nmol g1
Fig. 3. Active oxygen species (AOS) production by grapevine cell
suspensions after treatment with Pseudomonas spp. (A) Timecourse of H2O2 production in grapevine cell suspensions treated
with P. fluorescens CHA0 and P. aeruginosa 7NSK2 at 107 CFU
ml1, or in untreated cell suspensions (Ctrl). (B) Dose–response
curve of H2O2 production in grapevine cell suspensions, 85 min
after treatment with increasing concentrations of P. fluorescens
CHA0 and P. aeruginosa 7NSK2. (C, D) Maximum H2O2 produced
by grapevine cell suspensions, 85 min after treatment with P.
fluorescens CHA0, P. fluorescens WCS417, P. putida WCS358,
and P. fluorescens Q2-87 (C), and with P. aeruginosa 7NSK2 and
its mutants KMPCH and KPMCH-567 (D) at 107 CFU/ml, or in
untreated cell suspensions (Ctrl). Analyses were made on 5
independent batches of cell suspensions for each treatment.
Comparisons of statistical significance were made for each subfigure. Different letters indicate statistically significant differences
between treatments (Duncan’s multiple range test; P <0.05).
254 | Verhagen et al.
cells) occurred after treatment with CHA0 and Q2-87 as
DAPG producers. However, lower AOS production was
observed in response to WCS417 (Pvd+, Pch–, SA+).
WCS358 (Pvd+, Pch–, SA-) induced an intermediate level of
AOS similar to that provoked by 7NSK2 (about 40 nmol
g1 cells) (Fig. 3D). The mutant KMPCH (Pvd–, Pch–, SA+)
also triggered a high level of H2O2, and the effect of
KMPCH-567 strain (Pvd–, Pch–, SA–) resembled that of its
parental strain 7NSK2 (Fig. 3D). For all live strains no
oxidative burst occurred with a concentration of 106 CFU/
ml, and a threshold at 107 CFU ml1 was needed for the
induction of the H2O2 burst. Moreover, in all cases, the
treatment of the grape cells with the bacteria did not lead to
any increase in grapevine cell death during the time of the
experiments (data not shown).
Induction of the oxidative burst by bacterial cell extracts
and growing media
For a number of resistance-inducing bacteria, different
compounds including siderophores, lipopolysaccharide
(LPS), flagellin, and antibiotics (Duijff et al., 1997; Meziane
et al., 2005; Bakker et al., 2007) have been demonstrated to
act in a similar way as MAMPs (Jones and Dangl, 2006).
Recognition of these compounds leads to the activation of
inducible plant defence responses that may activate
resistance. The differential specificities of the selected
bacteria prompted an evaluation of the ability of bacterial
extracts and their end-growing medium to trigger an
oxidative burst. Results (Fig. 4A) show that the addition of
bacterial extracts of CHA0 and 7NSK2 to grape cell
suspensions triggered a strong oxidative burst within
minutes of treatment. The maximum amount of H2O2 has
already occurred at 30 min and reached approximately
60 lM H2O2 with both strains. The amount of H2O2
returned to the control level later on and no further increase
occurred. The time kinetic of the generation of AOS was the
same when grapevine cell suspensions were treated with the
growing medium (data not shown). The amount of H2O2
also attained a maximum after 30 min, but this was, in most
cases, lower than that induced by the bacterial extracts (Fig.
4B). These effects resembled those achieved by the MAMPs
(Aziz et al., 2004, 2007). The bacterial extracts of CHA0
and 7NSK2 showed similar dose–response characteristics
(Fig. 4C). A minimum initial concentration of bacteria of
106 CFU ml1 was required to cause a significant increase
in AOS, and the response was levelling off above 107
CFU ml1.
The bacterial extracts from WCS417, WCS358, Q2-87,
and derivative mutants of 7NSK2 also provoked an almost
immediate and strong burst of AOS, as exemplified by
extracts from CHA0 and 7NSK2 (Fig. 4A). The maximum
amounts of H2O2 produced by grape cells were comparable
for the extracts of WCS358, Q2-87, and CHA0 (around 560
nmol H2O2 g1 cells), but were statistically lower with
WCS417 (Pch–) (about 180 nmol H2O2 g1 cells) (Fig. 4D).
Extracts of KMPCH and KMPCH-567 mutants showed
similar responses to the extract of the wild-type
Fig. 4. Active oxygen species (AOS) production by grapevine cell
suspensions after treatment with crude cell extracts and growing
media of Pseudomonas spp. (A) Time-course of H2O2 production
in grapevine cell suspensions treated with crude cell extracts of
P. fluorescens CHA0 and P. aeruginosa 7NSK2 at an equivalent
concentration of live bacteria of 107 CFU ml1, or in untreated cell
suspensions (Ctrl). (B) Comparison of maximum amounts of AOS
produced after treatment of grapevine cell suspensions with living
bacteria, growing media, and cell extracts of P. fluorescens CHA0,
and P. aeruginosa 7NSK2. (C) Dose–response curve of AOS
production in grapevine cell suspensions, 30 min after treatment
with increasing concentrations of crude extracts of P. fluorescens
CHA0 and P. aeruginosa 7NSK2. (D, E) Maximum H2O2 produced
by grapevine cell suspensions, 30 min after treatment with cell
extracts of P. fluorescens CHA0, P. fluorescens WCS417,
P. putida WCS358, and P. fluorescens Q2-87 (D), and with
P. aeruginosa 7NSK2 and its mutants KMPCH and KPMCH-567
(E) at an equivalent concentration of live bacteria of 107 CFU ml1,
or in untreated cell suspensions (Ctrl). Analyses were made on five
independent batches of cell suspensions for each treatment.
Comparisons of statistical significance were made for each subfigure. Different letters indicate statistically significant differences
between treatments (Duncan’s multiple range test; P <0.05).
P. aeruginosa 7NSK2 (Fig. 4E). Although the timing was
identical to that observed with bacterial extracts (data not
shown), the H2O2 concentration was lower after treatment
of grape cell suspensions with the medium in which the
bacteria had grown for 24 h. The maximum amounts of
H2O2 differed between the bacterial strains. Medium from
Pseudomonas-mediated ISR in grapevine | 255
WCS417 and WCS358 cultures showed H2O2 amounts that
were slightly less than those produced in response to their
respective extracts. Treatment of grape cells with growing
medium from CHA0 or Q2-87 resulted in the production of
around 100 nmol g1 cells. This is about four times less
than with killed bacteria, but still twice more than with live
bacteria. Medium from the KMPCH and KMPCH-567
cultures showed similar AOS responses (about 60 nmol g1
cells) as that induced by the medium of 7NSK2 (Fig. 4B).
Phytoalexin accumulation in grapevine cells after
treatment with rhizobacteria
To investigate whether the resistance induced by selected
bacteria was associated with phytoalexin production, the
main grapevine stilbenes, resveratrol and e-viniferin, were
analysed in grape cells and leaves of plantlets treated with
live or killed bacteria. Control cells treated with MgSO4
exhibited a low level trans-resveratrol and its oligomer
trans-e-viniferin (less than 160.5 lg g1 fresh weight).
Nevertheless, large amounts of both phytoalexins were
produced by grapevine cells in response to most bacteria
(Fig. 5). Both resveratrol and viniferin accumulated during
the first hours and reached maximum amounts after 16 h of
treatment. Stilbene levels decreased later on, and returned
to basal levels after 48 h (Fig. 5A, B). High phytoalexin
responses were achieved with CHA0, Q2-87, and WCS358
(Fig. 5C). In response to P. fluorescens WCS417 viniferin
accumulated to a similar level (4–5 lg g1 fresh weight),
while it clearly induces less long-lasting resveratrol accumulation. The wild-type 7NSK2 and its mutants also led to an
accumulation of trans-resveratrol and trans-e-viniferin
(Fig. 5D) in grape cells. Both responses were 1.5–2-fold
lower with KMPCH and KMPCH-567, when compared to
the 7NSK2 strain.
Phytoalexin accumulation in grapevine cells after
treatment with bacterial extracts
The bacterial extracts that induced a strong oxidative burst
were also active in inducing a rapid accumulation of the
phytoalexins resveratrol and e-viniferin in grapevine cells
(Fig. 5E, F). Cell extracts of CHA0 and WCS417 were
almost as active in inducing resveratrol as live bacteria,
whereas the amounts of e-viniferin were less than after
treatment with live CHA0 (Fig. 5E). A significant resveratrol accumulation was observed in response to the
extracts of WCS358 and Q2-87, and viniferin accumulated
to a similar extent as induced by live bacteria. Extracts of
7NSK2 and its mutant KMPCH were also capable of
inducing a strong resveratrol and viniferin accumulation in
grapevine cells (Fig. 5F). However, KMPCH-567 induced
a 2-fold smaller accumulation of phytoalexins, but still
remained comparable to the live bacterium. These results
clearly show that bacterial extracts evoke comparable
responses in time and magnitude as live bacteria with
regard to phytoalexin production.
Fig. 5. Production of phytoalexins by grapevine cell suspensions
after treatment with living Pseudomonas spp. and their crude
extracts. (A, B) Time-course of trans-resveratrol (A) and transe-viniferin (B) production in grapevine cells treated with P.
fluorescens CHA0 and P. aeruginosa 7NSK2 at 107 CFU ml1, or
in untreated cells (Control). (C, D) Maximum phytoalexin amounts
in grapevine cells, 16 h after treatment with P. fluorescens CHA0,
P. fluorescens WCS417, P. putida WCS358 and P. fluorescens
Q2-87 (C), and with P. aeruginosa 7NSK2 and its mutants KMPCH
and KPMCH-567 (D) at 107 CFU ml1, or in untreated cells
(Control). (E, F) Maximum phytoalexin amounts in grapevine cells,
8 h after treatment with cell extracts of P. fluorescens CHA0, P.
fluorescens WCS417, P. putida WCS358, and P. fluorescens
Q2-87 (E), and with P. aeruginosa 7NSK2 and its mutants KMPCH
and KPMCH-567 (F) at an equivalent concentration of live bacteria
of 107 CFU ml1, or in untreated cells (Control). Analyses were
made on five independent batches of cell suspensions for each
treatment. Comparisons of statistical significance were made for
each sub-figure. Different letters indicate statistically significant
differences between treatments (Duncan’s multiple range test;
P <0.05).
Priming for enhanced phytoalexin production in leaf
tissues of treated plants
In order to investigate if the induced resistance against
B. cinerea is associated with the ability of bacteria to prime
phytoalexin production, roots of grapevine plantlets were
treated with selected bacteria or with their corresponding
extracts and leaves were subsequently removed and challenged with B. cinerea. In most cases, pretreatment of
grapevine plants with live bacteria and subsequent challenge
with B. cinerea led to significantly enhanced resveratrol and
256 | Verhagen et al.
viniferin accumulation when compared to the plants
pretreated with MgSO4 as the control (Fig. 6). While
CHA0, Q2-87, and WCS358 alone induce only small
amounts of resveratrol (10–25 lg g1 fresh weight) and
viniferin (15–30 lg g1 fresh weight) in grapevine leaves
before challenge, they act as inducers of priming for
enhanced phytoalexin production after challenge (Fig. 6A,
B). This effect is already apparent for resveratrol after 24 h
of challenge, and became more pronounced for the two
stilbenes at 3 d post-inoculation. This enhancement was
even stronger after 6 d of challenge (data not shown). By
contrast, the amounts of resveratrol and viniferin in the
plants pretreated with WCS417 are comparable to that of
control plantlets, where the amount of phytoalexins has also
increased as result of B. cinerea infection. These results are
not consistent with previous findings (Verhagen et al.,
2004), which demonstrated a phenomenon of gene expres-
sion priming in the P. fluorescens WCS417–Arabidopsis
system after challenge with Pst DC3000.
The priming activity of 7NSK2 and its mutants was also
evaluated in grapevine plantlets. Pretreatment with 7NSK2
resulted in a slight increase of phytoalexin levels before
challenge, but strongly primed resveratrol (Fig. 6C) and
e-viniferin (Fig. 6D) accumulation in the leaves after
challenge. Resveratrol and viniferin in the P. aeruginosa
7NSK2-treated plantlets reached a level of 2–3-fold when
compared with the control. The mutant P. aeruginosa
KMPCH also potentiated resveratrol and viniferin accumulation, but to a lower level than its wild type (Fig. 6C, D),
while P. aeruginosa KMPCH-567 had no consistent effect
on resveratrol production with 2 d of challenge. Nevertheless, the amounts of resveratrol and viniferin in the
KMPCH-567 treated plantlets was slightly enhanced after
3 d of B. cinerea inoculation when compared to the control.
Similar results are observed in detached leaves using
a short-term preincubation of 24 h with selected bacteria
(data not shown). Bacterial extracts also resulted in priming
similar phytoalexin responses as in grapevine cells (data not
shown).
Discussion
Fig. 6. Priming for phytoalexin production in leaf tissues of
grapevine plantlets by Pseudomonas spp. upon challenge with
Botrytis cinerea. Roots of grapevine plantlets were dipped in
bacterial suspension at 107 CFU ml1 and leaves were removed
and inoculated with B. cinerea. Analyses of phytolaexins were
conducted on leaves harvested before and during challenge. (A,
B) Time-course of primed trans-resveratrol (A) and trans-e-viniferin (B) production in leaf tissues of grapevine plants treated with
P. fluorescens CHA0, P. fluorescens WCS417, P. putida WCS358,
and P. fluorescens Q2-87 at 107 CFU ml1, or in untreated plants
(Control) upon challenge with B. cinerea. (C, D) Time-course of
primed trans-resveratrol (C) and trans-e-viniferin (D) production in
leaf tissues of grapevine plants treated with P. aeruginosa 7NSK2
and its mutants KMPCH and KPMCH-567 at 107 CFU ml1, or
in untreated plants (Control) upon challenge with B. cinerea.
For each treatment, analyses were made on 48 independent
plants. Comparisons of statistical significance were made for each
sub-figure. Different letters indicate statistically significant differences between treatments (Duncan’s multiple range test;
P <0.05).
Pseudomonas spp bacteria have been shown to trigger
induced systemic resistance (ISR) in different plant species.
This ISR is based on multiple mechanisms, including the
enhancement of the capacity of plants to mobilize cellular
defence responses before or upon pathogen challenge. In
this study, it is demonstrated for the first time that
P. fluorescens CHA0, P. fluorescens WCS417, P. putida
WCS358, P. fluorescens Q2-87, and P. aeruginosa 7NSK2
are all effective in inducing and/or priming defence
responses, and triggered resistance of grapevine against
B. cinerea. Treatment of detached grapevine leaves with live
rhizobacteria resulted in the significant control of
B. cinerea. The level of protection depends on the bacterial
strain, with WCS358 and 7NSK2 being the most active.
Comparable amounts of disease reduction were obtained
when bacteria were applied to the roots of grapevine
plantlets. Given the spatial separation of the challenging
leaf pathogen and the inducing rhizobacteria, these data
clearly indicate that disease protection resulted from an
induced state of resistance in grapevine plants. This is in
good agreement with the capability of triggering ISR in
other plants by P. fluorescens CHA0, P. fluorescens
WCS417, P. putida WCS358, and P. fluorescens Q2-87
(Pieterse et al., 1996; Iavicoli et al., 2003; Meziane et al.,
2005), and P. aeruginosa 7NSK2 (De Meyer et al., 1999;
Audenaert et al., 2002; De Vleesschauwer et al., 2006).
The effect of various strains, which were affected in the
expression of traits known to be operative in triggering ISR,
indicates that this induction is dependent on a specific
bacterium–grapevine interaction. This indicates an involvement of different bacterial compounds, but SA and the
SA-containing siderophore pyochelin did not seem to be
Pseudomonas-mediated ISR in grapevine | 257
necessary for triggering ISR by P. fluorescens WCS417 and
P. putida WCS358. For instance, P. fluorescens WCS417,
which is deficient in pyochelin, is effective in inducing
resistance in the leaves to a similar extent as CHA0.
Intriguingly, P. putida WCS358, which is defective in
pyochelin and SA, induces a strong leaf resistance against
B. cinerea, suggesting that pyoverdin or LPS must be
involved in triggering ISR. WCS358 was shown to produce
LPS and flagellin, which are all known to act as determinants of ISR in other plant species (Meziane et al., 2005;
Van Loon et al., 2008). In addition, LPS, siderophores, and
flagellin of P. putida WCS358 have been shown to be
involved in the induction of resistance in Arabidopsis, while
in bean and tomato only the LPS and siderophores
appeared to be active (Meziane et al., 2005). DAPG might
also be, in part, responsible for the induction of disease
resistance of grapevine leaves against B. cinerea, as indicated by the effect of P. fluorescens CHA0 and Q2-87.
CHA0, which is naturally present in the rhizosphere of
tobacco produced higher amount of DAPG (480–940 ng
108 CFU g1 of root) (Voisard et al., 1994). Similarly, in
Q2-87 that originates from wheat, the amount of DAPG
produced on roots is proportional to its rhizosphere
population density and reached approximately 44 ng per
107 CFU g1 of root (Raaijmakers et al., 1999). It has been
proposed that DAPG produced by P. fluorescens CHA0 is
detected by the roots of A. thaliana, in which it leads to
physiological changes that subsequently induce ISR in
leaves to Peronospora parasitica (Iaviocoli et al., 2003).
The experiments with detached leaves and P. aeruginosa
7NSK2 mutants showed that KMPCH, which is defective in
pyochelin and pyoverdin, enhanced local resistance against
B. cinerea equal to its parental strain, while KMPCH-567
that lacks pyoverdin, pyochelin, and SA production, was
not effective (Fig. 1). However, in ISR assays KMPCH
slightly compromised induced resistance, while KMPCH567 led to a significant loss of ISR against B. cinerea
(Fig. 2). This suggests that SA of P. aeruginosa 7NSK2 is
involved in triggering ISR in grapevine against B. cinerea as
previously reported in tomato, bean, and tobacco (De
Meyer et al., 1999; Audenaert et al., 2002). It is also
conceivable that the combined deficiency in pyochelin,
pyoverdin, and SA expression in KMPCH-567 could
contribute to the loss of grapevine resistance to B. cinerea.
Thus, resistance induced by P. aeruginosa 7NSK2 could
be attributed to the production of the pyocyanin (De
Vleesschauwer et al., 2006) or to a combined action of SA
or pyochelin and pyocyanin (Audenaert et al., 2002).
Our results also support the view that the resistance
induced was not dependent on the metabolic activity of
bacteria, since comparable amounts of resistance can be
induced with crude cell extracts. This is in accordance with
the production of heat-stable signalling molecules such as
LPS, flagellin, or secreted compounds, serving as inducers
of ISR (Bakker et al., 2007). The LPS seems to be a major
ISR-inducing determinant of P. fluorescens WCS417 in
radish and carnation (Van Wees et al., 1997), and of
P. putida WCS358 in tomato and bean against B. cinerea
(Meziane et al., 2005). LPS-containing cell walls also elicit
ISR in Arabidopsis (Van Wees et al., 1997; Meziane et al.,
2005).
Although P. aeruginosa KMPCH-567 provoked only
a partial disease reduction, its killed cells triggered a high
resistance against B. cinerea. These results suggest that some
compounds released after boiling bacteria could become
quantitatively sufficient to induce grape resistance. Effects
of the bacterial extracts, which contain cell-wall fragments
suggested also a possible role of membrane LPS in inducing
disease resistance (Van Loon et al., 2008).
In grapevine and other plants, the production of active
oxygen species (AOS) has been assumed to be one of the
earliest defence events in cell suspension cultures in response
to various MAMP elicitors (Aziz et al., 2007; Van Loon
et al., 2008; Varnier et al., 2009). It is shown here that live
bacteria triggered a rapid and long-lasting production of
AOS by grapevine suspension cells. Crude bacterial extracts
as well as culture media of selected bacteria all induced
a strong and transient oxidative burst lasting less than
30 min. In addition, the induced responses by live bacteria
or crude extracts (or media) are not primarily linked to
grapevine cell death (data not shown). Similar responses
have been reported in grapevine cells elicited by oligosaccharides (Aziz et al., 2004, 2007) or bacterial rhamnolipids
(Varnier et al., 2009), which also induced defence-related
gene expression and resistance in intact plants against
B. cinerea and Plasmopara viticola. These findings support
the view that H2O2 production in grape cell suspensions
could be predictive of the ability of P. fluorescens CHA0
and Q2-87 to induce ISR in whole plants. A positive
correlation was also observed between oxidative burst and
induced resistance against B. cinerea by P. putida WCS358
and P. aeruginosa 7NSK2, suggesting that H2O2 could
contribute as a signalling molecule for the induction of
disease resistance by these strains in planta. However, the
low elicitation of H2O2 in cell suspensions by WCS417
indicates that oxidative burst is not predictive of ISR in
grapevine plants. Inversely, in tobacco suspension cells
elicited with live WCS417, the early increase production of
H2O2 was not matched by the expression of ISR in whole
tobacco against Erwinia carotovora (Van Loon et al., 2008).
This is also the case for 7NSK2 mutants KMPCH and
KMPCH-567, which trigger a strong production of H2O2 in
grapevine cell suspensions, but showed only partial resistance against B. cinerea in whole plant.
Dose–response curves for H2O2 production showed
a threshold for live bacteria corresponding to 106 CFU g1
cells. However, for bacterial extracts as well as for growing
media, H2O2 production requires a minimum initial population density of live bacteria of 105 CFU g1 cells and reaches
a maximum at approximately 106 CFU g1 cells. This could
result either from an increased concentration of active
compounds in the medium, or from other beneficial
microbe-derived MAMPs such as LPS. These results are in
agreement with those of Van Loon et al. (2008) showing that
a threshold for cell wall preparations of 105 CFU g1 cells
was required for the oxidative burst in cell suspensions of
258 | Verhagen et al.
tobacco and the response reached its maximum at 106 CFU
g1 cells. This is consistent with an early finding showing that
the induction of systemic resistance is a threshold-dependent
mechanism (Raaijmakers et al., 1995).
The growing medium of P. fluorescens WCS417 and
P. putida WCS358 led to a higher oxidative burst when
compared with P. fluorescens CHA0 and Q2-87, and
P. aeruginosa 7NSK2. The first two bacteria have been
shown to trigger resistance with LPS, flagellin, and siderophores, while the latter use siderophores and DAPG
(Bakker et al., 2007). This could indicate that in the culture
medium of the first two bacteria multiple MAMPs are
present, leading to a higher oxidative burst, or that the LPS
or flagellin are stronger inducers of the oxidative burst as
reported in tobacco cell suspensions (Van Loon et al., 2008).
Moreover, cell extracts and growing media of P. aeruginosa
KMPCH and KMPCH-567 showed similar AOS responses
as those of the wild-type 7NSK2. This suggests that, rather
than siderophores, other elements secreted by P. aeruginosa
7NSK2 could be involved in inducing the oxidative burst.
P. aeruginosa 7NSK2 produced the antibiotic pyocyanin that
generated H2O2 and triggered ISR in rice plants against
Magnaporthe grisea (De Vleesschauwer et al., 2006) or in
tomato against B. cinerea (Audenaert et al., 2002). Further
experiments are needed to investigate the exact determinants
that induce the oxidative burst in our system.
Most of rhizobacteria also induced a small and transient
increase in the amount of stilbene phytoalexins, transresveratrol and its dimer e-viniferin, in grapevine suspension
cells and leaves. Both phytoalexins have been shown to
possess biological activity against a wide range of pathogens
and can be considered as markers for plant disease
resistance (Langcake, 1981; Coutos-Thevenot et al., 2001;
Jeandet et al., 2002). As for the oxidative burst, the lower
response was observed with P. fluorescens WCS417. In
response to viable cells or extracts of P. fluorescens CHA0
and Q2-87, and P. putida WCS358, phytoalexin accumulation is correlated to the amount of the oxidative burst as for
cell extracts of 7NSK2 and KMPCH. However, live
P. aeruginosa KMPCH and KMPCH-567, which triggered
a strong AOS burst induced only small amounts of
phytoalexins. These results support the view that CHA0
and 7NSK2 bacteria use distinct signalling pathways to
trigger ISR and suggest that grapevine is endowed with
multiple inductive resistance pathways. This could, at least
partly, explain the discrepancies between the level of
induced resistance, oxidative burst, and phytoalexin
responses.
According to the priming concept (Conrath et al., 2002),
the enhanced phytoalexin accumulation in grapevine leaves
after root treatments with P. fluorescens CHA0, Q2-87 and
P. putida CWS358, or their corresponding extracts suggests
a pathogen-dependent activation of defence responses in
grapevine plants. By contrast, P. fluorescens WCS417,
which induced ISR, did not primed phytoalexins. This
suggests the co-existence of multiple pathways in grapevine
leading to induced resistance against B. cinerea. P. aeruginosa
7NSK2 also efficiently primed grapevine plants to accumu-
late resveratrol and viniferin upon pathogen attack, resulting in enhanced resistance to B. cinerea. In line with this
concept, De Vleesschauwer et al. (2006) previously uncovered priming as a crucial facet of the resistance
mechanism underlying P. aeruginosa 7NSK2-mediated ISR
against M. oryzae. The mutant KMPCH, which induced
a partial resistance, also potentiated slight amounts of
resveratrol and viniferin after challenge, while the
KMPCH-567 strain had no consistent effect. This suggests
that, besides SA, pyochelin and/or pyoverdin are important
in priming phytoalexin response and induced resistance in
grapevine by 7NSK2. This is consistent with a recent study
by De VIeesschauwer et al. (2008) showing that pseudobactin (pyoverdin) from P. fluorescens WCS374 primed for
a SA-repressible defence response and elicited ISR against
Magnaporthe oryzae. To our knowledge, in grapevine it is
the first time that rhizobacteria have been found to prime
defence reactions. Priming has also been previously
reported for BABA- and b-1.3-glucan sulfate-induced
resistance in grapevine against Plasmopara viticola (Hamiduzzaman et al., 2005; Trouvelot et al., 2008). In the light of
these results, a pathogen-dependent activation of defence
mechanisms appears to be a common feature of induced
resistance in grapevine.
In summary, these results clearly demonstrated that
treatment of grapevine cells or roots by Pseudomonas spp.
and their cellular extracts induced and/or primed the
expression of cellular defence responses, resulting in an
enhanced level of induced resistance against B. cinerea. Our
results also provide some evidence for the implication of
siderophore pyoverdin or LPS, rather than SA and pyochelin in triggering ISR by P. fluorescens WCS417 and
P. putida WCS358, while the antibiotic DAPG seems to be
a major ISR determinant in P. fluorescens CHA0 and Q287. However, it is evident from the present study that SA,
pyochelin, and pyoverdin are important in priming defence
responses and triggering ISR by P. aeruginosa 7NSK2. This
also suggests the co-existence of different pathways in
grapevine leading to induced resistance against B. cinerea.
Further elucidations are needed to understand the mechanisms underlying rhizobacteria-induced priming of defence
reactions as related to systemic resistance in grapevine.
Acknowledgements
We are grateful to Professor Dr CMJ Pieterse for providing
P. fluorescens WCS417 and P. putida WCS358. P. fluorescens CHA0 was a kind gift from Professor Dr D Haas. We
are also grateful to Professor Dr DM Weller for providing
P. fluorescens Q2-87. This research was supported by
Champagne-Ardenne Region and Europol’Agro through
the VINEAL programme.
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