Pseudomonas spp.-induced systemic resistance to Botrytis cinerea is associated with induction

Journal of Experimental Botany, Vol. 61, No. 1, pp. 249–260, 2010doi:10.1093/jxb/erp295 Advance Access publication 7 October, 2009

RESEARCH PAPER

Pseudomonas spp.-induced systemic resistance to Botrytiscinerea is associated with induction and priming of defenceresponses in grapevine

Bas W. M. Verhagen1,*, Patricia Trotel-Aziz1, Michel Couderchet1, Monica Hofte2 and Aziz Aziz1,†

1 URVVC – Stress & Environment EA 2069, PPDD, University of Reims, F-51687 Reims cedex 2, France2 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 enhancedoxidative 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) andKMPCH-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 rhizobacteria-

induced 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]: �AOS, active oxygen species; DAPG, 2;4-diacetylphloroglucinol; ISR, induced systemic resistance; LPS, lipopolysaccharides; MAMPs, microbe-associated 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]

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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 theinduced 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 beenobserved 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; Magnin-

Robert 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 patho-

gen attack. This enhanced capacity to express infection-

induced 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 recogni-tion 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, andflagellin 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 theonly 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

Hofte, 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 by7NSK2 (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; Trotel-

Aziz et al., 2008). Nevertheless, grapevine plants can express

various defence mechanisms during interaction with patho-

genic micro-organisms (Busam et al., 1997; Bezier 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 accumula-

tion 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 isone 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 expres-

sion 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 atten-tion 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; Delau-

nois 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-O-

b-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 grapevineresistance 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 Pvd-deficient) and KMPCH-567 (Pch-, Pvd-, and SA-deficient).

The effects of crude bacterial extracts or growing medium

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were also evaluated and some defence responses, such as the

production of active oxygen species (AOS) and phytoalexinaccumulation, 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 Table1. Pseudomonas fluorescens CHA0, WCS417, Q2-87, P. putidaWCS358, P. aeruginosa 7NSK2 and its mutants KMPCH andKMPCH-567 bacteria were grown in liquid Luria-Bertani (LB) orKing B (KB) medium (King et al., 1954), for 24 h at 28 �C and 120rpm as described previously (Pieterse et al., 1996). Bacteria werecollected by centrifugation and resuspended in 10 mM MgSO4 at105, 106, and 107 CFU ml�1. To check the effects of the medium inwhich bacteria have grown, the medium fraction was collected asa supernatant after centrifugation of each suspension of livebacteria. The remaining pellet containing bacteria were resus-pended in MgSO4 and boiled at 95 �C for 15 min to obtainedcrude cell extracts from different initial concentrations of livebacteria. Both bacterial extracts and media were checked for theabsence of bacterial cells by plating them onto LB or KB agarplates.

Fungal pathogen

Botrytis cinerea strain 630 was cultured in Erlenmeyer flaskscontaining potato dextrose agar (Sigma) for 4 weeks at 22 �C.Spores were collected and resuspended in sterile distilled water toa final density of 106 conidia ml�1. The spores were usedimmediately 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) wereobtained in vitro by multiplication through in vitro micro-cuttingson modified Murashige and Skoog (1962) medium, as describedpreviously (Aziz et al., 2003). Plantlets were grown at 24 �C with16/8 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 incontinuous light. Cells were maintained in the exponential phaseand subcultured 24 h prior to use.

Protection assays on detached leaves

Leaves were excised from 10-week-old in vitro-grown grapevineplantlets and treated by floating them, adaxial side downward, onstandard buffer (2 mM MES, pH 5.9, containing 0.5 mM CaCl2and 0.5 mM K2SO4), containing live bacteria or their correspond-ing extracts or media, with equivalent initial concentrations of livebacteria. After 48 h, leaves were rinsed with distilled water andplaced on wet absorbing paper in glass Petri dishes. One needle-prick wound was applied to the abaxial side of each leaf andcovered with 7.5 ll drops of a conidial suspension of B. cinerea(106 conidia ml�1). Quantification of disease development wasmeasured as the average diameter of lesions formed at 5 d post-inoculation, and the average disease in the control was used tocalculate the % of disease reduction.

Induced systemic resistance

Four-week-old grapevine plantlets were uprooted and treated bydipping the root system in a 20 ml suspension of live bacteria (107

CFU ml�1) or corresponding cell extracts in 10 mM MgSO4 for15 s before transplanting into a new medium of Murashige andSkoog (1962). After 15 d or 28 d, the leaves were detached fromtreated and control plantlets and challenge inoculated withB. cinerea as described before. The average lesion size wasdetermined after 5 d, and the % disease reduction was calculatedby comparing the treatments with the average lesion size in thechallenged control.

Treatment of cell suspensions

Cells were collected during the exponential growth phase andwashed by filtration in a suspension buffer containing 175 mMmannitol, 0.5 mM K2SO4, 0.5 mM CaCl2, and 2 mM MES, pH5.5. Cells were resuspended at 0.1 g FW ml�1 with suspensionbuffer 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 tolive bacteria. Control cells were incubated under the sameconditions and treated with MgSO4 solution. Grapevine cells werethen used for measurements of H2O2 production, phytoalexinaccumulation, and cell death during treatments.

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+

Voisard et al., 1994; Iavicoli et al., 2003

Pseudomonas fluorescens WCS417 Isolated from wheat rhizosphere; OA+,

Pvd+, Pch-, SA+

Lamers et al., 1988; Leeman et al., 1995

Pseudomonas fluorescens Q2-87 Isolated from wheat roots; DAPG+ Raaijmakers et al., 1997

Pseudomonas putida WCS358 Isolated from potato rhizosphere; OA+, Pvd+,

Pch-, SA-

Geels and Schippers, 1983; Van Vees et al., 1997

Pseudomonas aeruginosa 7NSK2 Isolated from bean rhizosphere; Pvd+, Pch+,

SA+, Pyo+,

Iswandi et al., 1987

Pseudomonas aeruginosa KMPCH Pyo+, Pvd–, Pch–, SA+, chemical mutant of the

pyoverdine-negative mutant MPFM1, Kmr

Hofte et al., 1993

Pseudomonas aeruginosa KMPCH-567 Pvd–, Pch–, SA–, pchA replacement mutant of

KMPCH; Kmr

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.

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H2O2 production

The production of H2O2 by the cells was assayed by chemilumi-nescence using luminol as described in Aziz et al. (2003). Cells weretreated and chemiluminescence was measured in a 10 s period witha luminometer (Lumat LB 9507, Berthold).

Cell viability was assayed using the vital dye Neutral Red asdescribed in Aziz et al. (2003).

Phytoalexin quantification

The isolation and analysis of phytoalexins in cell cultures wasperformed as described by Aziz et al. (2003). Stilbenes from thecells (2 ml) were extracted in 2 ml of 85% methanol overnight at4 �C. After centrifugation (10 min, 5000 g), supernatants wereevaporated under nitrogen and the samples were solubilized in1 ml of 100% methanol. Phytoalexins were analysed by HPLCusing a linear gradient of 10–85% acetonitrile at a flow rate of 1 mlmin�1. Phytoalexins, trans-resveratrol and e-viniferin weredetected with a photodiode array detector coupled to a fluorometerdetector (kex ¼ 330 nm, kem ¼ 374 nm) and quantified on thebasis of standard calibration curves. The isolation and analysis ofphytoalexins from leaves follow the same procedures with slightmodifications as described by Aziz et al. (2006).

Statistical analysis

All experiments were done at least five times per treatment andstatistical analysis was performed by analysis of variance andDuncan’s multiple range test, using SigmaStat 3.5 Systat Software.Comparisons of statistical significance were made for eachsub-figure.

Results

Reduction of B. cinerea development in detachedleaves

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 grape-vine, 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 bacte-rium, 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 toinsufficient colonization of the medium, because population

densities of 7NSK2 and its mutants are similar (1–1.23107

CFU ml�1).

Crude cell extracts of bacteria were also used in

protection assays. As can be seen in Fig. 1C, D, most of

the bacterial extracts were capable of reducing necroticlesions 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 reducingdisease.

Enhanced disease resistance in the leaves of treatedplants to B. cinerea

To rule out the possibility that the observed disease

protection was due to direct effects of bacteria onB. 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

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 ml�1, 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 ml�1, 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 treat-

ments (Duncan’s multiple range test; P <0.05).


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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 preparationswere 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 cellsuspensions by rhizobacteria

To investigate if the oxidative burst might represent aninitial 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

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 ml�1 corresponding to 105 CFU g�1

grape cells, while a threshold for live cells of both strains at

107 CFU ml�1 (equivalent to 106 CFU g�1 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 factorsknown 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. fluores-

cens 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 g�1

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 ml�1, 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 ml�1, 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).

Fig. 3. Active oxygen species (AOS) production by grapevine cell

suspensions after treatment with Pseudomonas spp. (A) Time-

course of H2O2 production in grapevine cell suspensions treated

with P. fluorescens CHA0 and P. aeruginosa 7NSK2 at 107 CFU

ml�1, 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 sub-

figure. Different letters indicate statistically significant differences

between treatments (Duncan’s multiple range test; P <0.05).


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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

g�1 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 itsparental 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 ml�1 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 extractsand 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 anoxidative 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 thesame 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 of106 CFU ml�1 was required to cause a significant increase

in AOS, and the response was levelling off above 107

CFU ml�1.

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 comparablefor the extracts of WCS358, Q2-87, and CHA0 (around 560

nmol H2O2 g�1 cells), but were statistically lower with

WCS417 (Pch–) (about 180 nmol H2O2 g�1 cells) (Fig. 4D).

Extracts of KMPCH and KMPCH-567 mutants showed

similar responses to the extract of the wild-type

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

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 ml�1, 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 ml�1,

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 sub-

figure. Different letters indicate statistically significant differences

between treatments (Duncan’s multiple range test; P <0.05).


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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 g�1 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 g�1

cells) as that induced by the medium of 7NSK2 (Fig. 4B).

Phytoalexin accumulation in grapevine cells aftertreatment with rhizobacteria

To investigate whether the resistance induced by selectedbacteria 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 g�1 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 g�1 fresh weight),

while it clearly induces less long-lasting resveratrol accumu-lation. 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 aftertreatment 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 resvera-

trol 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 induceda 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.

Priming for enhanced phytoalexin production in leaftissues 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 weretreated with selected bacteria or with their corresponding

extracts and leaves were subsequently removed and chal-

lenged 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

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 trans-

e-viniferin (B) production in grapevine cells treated with P.

fluorescens CHA0 and P. aeruginosa 7NSK2 at 107 CFU ml�1, 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 ml�1, 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 ml�1, 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).


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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 g�1 fresh weight) and

viniferin (15–30 lg g�1 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 hof 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 accumu-

lation, 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. Neverthe-

less, the amounts of resveratrol and viniferin in the

KMPCH-567 treated plantlets was slightly enhanced after3 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

Pseudomonas spp bacteria have been shown to trigger

induced systemic resistance (ISR) in different plant species.

This ISR is based on multiple mechanisms, including theenhancement 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 liverhizobacteria 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 aninduced 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 theexpression of traits known to be operative in triggering ISR,

indicates that this induction is dependent on a specific

bacterium–grapevine interaction. This indicates an involve-

ment of different bacterial compounds, but SA and the

SA-containing siderophore pyochelin did not seem to be

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 ml�1 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-vin-

iferin (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 ml�1, 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 ml�1, 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 differ-

ences between treatments (Duncan’s multiple range test;

P <0.05).


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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 produceLPS and flagellin, which are all known to act as determi-

nants 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 diseaseresistance of grapevine leaves against B. cinerea, as in-

dicated 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

10�8 CFU g�1 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 per107 CFU g�1 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 againstB. 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 KMPCH-

567 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 (DeMeyer 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 inradish 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. Effectsof 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 livebacteria 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 oligosac-charides (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 andinduced 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 ofH2O2 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 re-

sistance against B. cinerea in whole plant.

Dose–response curves for H2O2 production showed

a threshold for live bacteria corresponding to 106 CFU g�1

cells. However, for bacterial extracts as well as for growing

media, H2O2 production requires a minimum initial popula-

tion density of live bacteria of 105 CFU g�1 cells and reaches

a maximum at approximately 106 CFU g�1 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 thata threshold for cell wall preparations of 105 CFU g�1 cells

was required for the oxidative burst in cell suspensions of


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tobacco and the response reached its maximum at 106 CFU

g�1 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 beenshown to trigger resistance with LPS, flagellin, and side-

rophores, 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). Furtherexperiments 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, trans-

resveratrol 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 diseaseresistance (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 accumula-

tion 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 triggereda 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 grapevineleading to induced resistance against B. cinerea. P. aeruginosa

7NSK2 also efficiently primed grapevine plants to accumu-

late resveratrol and viniferin upon pathogen attack, result-

ing in enhanced resistance to B. cinerea. In line with this

concept, De Vleesschauwer et al. (2006) previously un-

covered 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 theKMPCH-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 pseudobac-

tin (pyoverdin) from P. fluorescens WCS374 primed for

a SA-repressible defence response and elicited ISR against

Magnaporthe oryzae. To our knowledge, in grapevine it isthe 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 (Hami-

duzzaman 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 pyoche-

lin in triggering ISR by P. fluorescens WCS417 andP. putida WCS358, while the antibiotic DAPG seems to be

a major ISR determinant in P. fluorescens CHA0 and Q2-

87. 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 mecha-nisms 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. fluores-

cens 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 throughthe VINEAL programme.

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Pseudomonas spp.-induced systemic resistance to Botrytis cinerea is associated with induction

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