APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2006, p. 7246–7252 Vol. 72, No.

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0099-2240/06/$08.00⫹0 doi:10.1128/AEM.01047-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Enhancement of Chilling Resistance of Inoculated Grapevine

Plantlets with a Plant Growth-Promoting Rhizobacterium,
Burkholderia phytofirmans Strain PsJN䌤
Essaid Ait Barka,1* Jerzy Nowak,2 and Christophe Clément1
Laboratoire de Stress, Défenses et Reproduction des Plantes, Unité de Recherche Vignes et Vins de Champagne, UPRES EA 2069,
UFR Sciences, Université de Reims Champagne-Ardenne, 51687 Reims Cédex 2, France,1 and Department of Horticulture,
Virginia Polytechnic Institute and State University, 0327-301 Saunders Hall, Blacksburg, Virginia 240602
Received 5 May 2006/Accepted 7 September 2006

In vitro inoculation of Vitis vinifera L. cv. Chardonnay explants with a plant growth-promoting rhizobacte-
rium, Burkholderia phytofirmans strain PsJN, increased grapevine growth and physiological activity at a low
temperature. There was a relationship between endophytic bacterial colonization of the grapevine plantlets and
their growth at both ambient (26°C) and low (4°C) temperatures and their sensitivities to chilling. The major
benefits of bacterization were observed on root growth (11.8- and 10.7-fold increases at 26°C and 4°C,
respectively) and plantlet biomass (6- and 2.2-fold increases at 26°C and 4°C, respectively). The inoculation
with PsJN also significantly improved plantlet cold tolerance compared to that of the nonbacterized control.
In nonchilled plantlets, bacterization enhanced CO2 fixation and O2 evolution 1.3 and 2.2 times, respectively.
The nonbacterized controls were more sensitive to exposure to low temperatures than were the bacterized
plantlets, as indicated by several measured parameters. Moreover, relative to the noninoculated controls,
bacterized plantlets had significantly increased levels of starch, proline, and phenolics. These increases
correlated with the enhancement of cold tolerance of the grapevine plantlets. In summary, B. phytofirmans
strain PsJN inoculation stimulates grapevine growth and improves its ability to withstand cold stress.

Abiotic stress leads to a series of molecular, biochemical,
physiological, and morphological changes that adversely affect
plant growth and productivity. A low temperature is a major
factor limiting the productivity and geographical distribution
of many species, including important agricultural crops (3).
Cold-hardy plants increase their freezing tolerance upon ex-
posure to low, nonfreezing temperatures by a phenomenon
known as cold acclimation (38).
The process of cold acclimation involves changes in gene
expression profiles (32), membrane lipids, total protein con-
tent, and composition of soluble proteins (30). Increases in the
activity of oxygen-scavenging enzymes (11), sugar, and proline
content (2, 3, 41), anthocyanin accumulation, and altered
growth morphology (30) have also been reported. Moreover,
some plants produce specific proteins that protect cells under
supercooling conditions, hence lowering the freezing point (6).
These, among other changes, alter leaf ultrastructure (28) and
modify plant growth patterns, affecting morphology (32).
Epiphytic bacterial species with ice-nucleating activity (Ice⫹
bacteria), such as Pseudomonas syringae, contribute to the frost
injuries of many cold-sensitive plants by reducing the plants’
ability to supercool, a process that prevents the formation of
membrane-damaging ice crystals (21). Since the ice nucleation
temperature increases with an increasing population size of
Ice⫹ bacteria, preemptive competitive exclusion of Ice⫹ bac-
* Corresponding author. Mailing address: Laboratoire de Stress,
Défenses et Reproduction des Plantes, Unité de Recherche Vignes et
Vins de Champagne, UPRES EA 2069, UFR Sciences, Université de
Reims Champagne-Ardenne, 51687 Reims Cédex 2, France. Phone
and fax: 33-3-26-91-34-41. E-mail: ea.barka@univ-reims.fr.
䌤
Published ahead of print on 15 September 2006.
teria by naturally occurring but non-ice-nucleating active bac-
teria could be an effective and practical means of frost control
in cold-sensitive plants (21). Recently, commercial formula-
tions combining bacteria antagonistic to plant pathogenic mi-
crobes with ice nucleation-active bacteria have been utilized as
an environmentally safe method to manage biotic and abiotic
stress in plants (21). In addition, some of these bacteria, such
as epiphytic or endophytic plant growth-promoting rhizobac-
teria (PGPR), enhance plant growth while improving their
resistance to stress (10, 13, 15, 22, 26). PGPR can stimulate
developmental changes in host plants (13, 17), disrupt phyto-
pathogen organization (4, 5), induce systemic resistance to
pathogens (7, 8, 13, 34), affect phytohormone production, and
improve nutrient and water management (13, 25). In vitro-
bacterized plantlets not only grew faster than nonbacterized
controls but also were sturdier, with a better-developed root
system (4) and significantly greater capacities for withstanding
biotic (4, 5, 34) and abiotic (9) stresses.
In this study, we tested a known plant growth-promoting
bacterium, Burkholderia phytofirmans strain PsJN, for its ability
to enhance chilling resistance in grapes. This bacterium is
capable of epiphytic and endophytic colonization of grapevine
tissues and organs (4, 12) and has been shown to protect plants
against heat stress (9). Our hypothesis was that endophytic
colonization of grapevines by Burkholderia phytofirmans strain
PsJN would cause physiological and biochemical changes in
the plants that may enhance their cold tolerances upon expo-
sure to low temperatures. To address this hypothesis, we ex-
amined the levels of electrolytes, proline, phenolics, and starch
content and related them to gas exchange and chilling toler-
ance in bacterized and nonbacterized plantlets under cold

7246
VOL. 72, 2006 B. PHYTOFIRMANS IMPROVES GRAPEVINE COLD TOLERANCE
FIG. 1. Effect of chilling injury on the weight of grapevine plantlets. (A) Nonchilled plantlets. (B) Chilled plantlets. Error bars indicate standard
deviations.
stress. Morphologies and cytologies of grapevine organs before
and after chilling were also examined.
Vineyards north of Reims (Champagne) are from 48° to
49°5⬘ latitude and occur at the northern limit of the area where
grapes are grown for production in France. Nonfreezing but
cool temperatures are common during the growing season in
this area and can compromise grapevine productivity. Since
grapes are clonally propagated, PGPR could be established in
planta during in vitro propagation and potentially provide a
carryover effect of enhancing chilling stress resistance in viti-
culture.
MATERIALS AND METHODS
Plant material and in vitro growth conditions. Plantlets of Vitis vinifera L. cv.
Chardonnay clone 7535 were micropropagated by nodal explants grown on 15 ml
of semisolid medium (23) in 25-mm culture tubes under 200 ␮mol m⫺2 s⫺1 white
fluorescent light for a 16-h photoperiod at 26°C (12). Uniform plantlets (n ⫽ 24)
were selected for each treatment in each experiment.
Plant bacterization. The bacterial inoculum was produced by transferring two
loops of PsJN to 100 ml of King’s B liquid medium in a 250-ml Erlenmeyer flask
incubated at 20°C at 150 rpm for 48 h (4). Bacteria were collected by centrifu-
gation (3,000 ⫻ g for 15 min) and washed twice with phosphate-buffered saline
(PBS) (10 mM, pH 6.5). The pellet was resuspended in PBS and used as inoc-
ulum. The bacterial concentration was estimated by spectrophotometry (600 nm)
and adjusted to 106 CFU/ml with PBS (5). The concentration was confirmed by
plate counting. Nodal explants, approximately 1 cm long and taken from 6-week-
old stock plantlets, were immersed in the inoculum for 1 min, blotted with sterile
filter paper, and placed in culture tubes as described previously (5). Noninocu-
lated control plantlets were dipped in PBS. The plants were incubated in the
growth chamber as described above.
Cold treatment. After 6 and 12 weeks, bacterized and nonbacterized grapevine
plantlets were divided into two groups: one group was transferred to a cold
growth chamber maintained at 4°C under a 16 h-photoperiod with light provided
by white fluorescent lamps at an intensity of 200 ␮mol m⫺2 s⫺1, and the other
(control) group was kept under the conditions described above. Each treatment
consisted of 24 plantlets, and the experiment was repeated three times. Analyses
were conducted after 2 weeks of treatment.
Electrolyte leakage. Leaf samples comprising the fifth and sixth leaves from
the basal end of the plantlets were taken from each plantlet (n ⫽ 24), rinsed with
7247
distilled water, and dried on filter paper. The leaves were incubated in 30 ml
mannitol (0.4 M) in 50-ml plastic bottles at 24°C for 20 h on a rotary shaker (80
rpm) as described previously (18). The conductance of the incubation medium
was measured using a conductivity meter (Orion, model 150; Thermo Electron
Corporation, Breda, The Netherlands). Samples were autoclaved at 120°C for 3
min and cooled to room temperature, and the volumes were adjusted to 30 ml.
The conductivity of the samples was measured again to determine the total
electrolyte content of the tissue. The degree of electrolyte leakage was calculated
as described earlier (1).
Free proline analysis. Free proline content was determined as described by Ait
Barka and Audran (3). Plantlets were frozen in liquid N2 and kept at ⫺80°C until
used. Leaves, stems, and roots were ground separately and homogenized in 3%
(wt/vol) sulfosalicylic acid. The homogenate was filtered through filter paper
(Whatman no. 1). After the addition of ninhydrin reagent (1% [wt/vol] ninhydrin
in 60% acetic acid), the mixture was heated to 100°C for 20 min. The reaction
was then stopped in ice. The mixture was extracted with 1 ml toluene, and the
sample was vigorously shaken for 15 s. After 4 h in darkness at room tempera-
ture, sample absorbance of the toluene layer was read at 520 nm. Proline
concentration was determined by using a calibration curve and expressed as ␮M
proline g⫺1 fresh weight (FW).
Determination of total phenolics. The content of total phenolics was deter-
mined by using a modified Folin-Ciocalteu colorimetric method (36). Fresh leaf
tissue (600 mg) was ground in 5 ml ethanol (80%) using a tissue homogenizer.
Samples were placed in 50-ml tightly covered plastic tubes and incubated at 4°C
for 2 h in the dark and then filtered as above. The pellet was resuspended in 2.5
ml ethanol. Five replicates of 125 ␮l phenolic extract, 625 ␮l 1/10 diluted Folin-
Ciocalteu reagent, and 250 ␮l 7.5% (wt/vol) Na2CO3 were vortexed for 10 s, and
the mixture was incubated at 45°C in a water bath shaker for 15 min. Phenolics
were measured at 750 nm using catechin as the standard. Total phenolics were
expressed as ng g⫺1 FW.
Gas exchange measurements. Carbon dioxide exchange rates were measured
on 24 plantlets from each treatment using a Li-Cor 6200 portable photosynthetic
system (Li-Cor, Lincoln, Neb.) and a 250-ml gas exchange chamber. During the
measurement, the gas exchange chamber was illuminated with a white light
source (1,000 ␮mol photons · m⫺2 · s⫺1) under a relative humidity of 65 to 75%,
an air CO2 concentration of 420 to 460 ␮l liter⫺1, and a flow rate of 1,125 ␮mol ·
min⫺1. O2 evolution was measured with an oxygen electrode (Hansatech, Cam-
bridge, United Kingdom). Three leaves were detached from each plant and
placed in the electrode chamber. Saturating CO2 conditions were maintained
using 2 M potassium carbonate-potassium bicarbonate buffer, pH 9.3. The elec-
trode buffer contained saturated potassium chloride.
7248 AIT BARKA ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 1. Effect of chilling injury on electrolyte leakage from leaves, leaf-free proline, total phenolics, and starch content of grapevine plantletsa
Electrolyte Free proline (␮moles g⫺1 FW) Starch content (mg/g of FW)
Total phenols
Treatment leakageb
(nanogram g⫺1 FW)
(%) Shoots Leaves Roots Shoots Leaves Roots

Control at 26°C 13.00 a 0.95 a 2.85 a ND a 13.11 a 0.005 a 0.010 a 0.010 a

Control at 4°C 39.00 b 1.89 b 5.75 b 0.09 a 14.51 b 0.005 a 0.010 a 0.002 a
Bacterized at 26°C 14.00 a 1.24 c 3.5 c 0.15 b 19.85 c 0.060 b 0.050 b 0.004 a
Bacterized at 4°C 19.95 c 2.19 d 6.45 d 0.18 b 21.55 d 0.050 c 0.080 c 0.002 a
a
Values correspond to the mean obtained with 24 plantlets for each replicate. Three replicates were performed per treatment. In each row, the values represented
by the same lowercase letters were not significantly different at a P value of ⬍0.05 (Fisher’s protected LSD test). ND, not detected.
b
Expressed as percentage of total electrolyte content.

Starch extraction and analysis. Organs were sampled from each plantlet (n ⫽
24) and homogenized individually at 4°C in a mortar containing 0.1 M phosphate
buffer, pH 7.5. The homogenates were centrifuged at 12,000 ⫻ g for 15 min, and
the pellets were used for starch analysis. The collected pellets were resuspended
in dimethyl sulfoxide-8 M hydrochloric acid (4:1, vol/vol). Starch was dissolved
over 30 min at 60°C with agitation (60 rpm). After centrifugation for 15 min at
12,000 ⫻ g, 100-␮l supernatant samples were mixed with 100 ␮l iodine-HCl
solution (0.06% KI and 0.003% I2 in 0.05 M HCl) and 1 ml distilled water. The
absorbance was read at 600 nm after 15 min of incubation at room temperature.
Microscopic observations. Roots, stems, and leaves were cut into 1-mm sec-
tions. The samples were immersed in cold fixative solution containing 8% glu-
taraldehyde, 2% paraformaldehyde in 0.2 M potassium buffer (pH 7.24), vacuum
infiltered for 20 min, and immersed in fresh fixative solution for 20 h (18).
Samples were subsequently washed with 0.2 M potassium buffer (pH 7.24),
postfixed in 2% osmium tetroxide prepared in the same buffer for 4 h, washed
with the buffer, and dehydrated in a graded ethanol series. The samples were
then washed with acetone series and embedded in araldite (Fluka, France).
Semithin sections (1 ␮m) were collected on glass slides and stained with toluidine
blue, rinsed in distilled water, air dried, and mounted in Eukitt. The sections
were examined under an optical microscope (model no. BH-2; Olympus, Tokyo,
Japan).
Statistical analyses. Unless stated otherwise, replicates of 24 plants were used
per treatment. Collected data were analyzed statistically using analysis of vari-
ance. Means for each treatment were separated with at least significant differ-
ence (LSD; P ⬍ 0.05) multiple comparison test (Fisher’s protected). All exper-
iments were repeated three times.
RESULTS
Under the applied experimental conditions, the changes in
all analyzed parameters were similar for both 6- and 12-week-
old plantlets. The results of the experiments conducted on
12-week-old plantlets are presented.
Plant bacterization. A comprehensive PsJN colonization
study of grapevine plantlets was completed earlier using a
PsJN::gfp2X derivative tagged with green fluorescent protein.
We found that the bacterium initially colonized the root sur-
face, followed by tissue penetration and colonization of the
root interior, translocation via stem xylem vessels, and then
endophytic colonization of leaf tissues (12). A study conducted
under the same in vitro culture and bacterial inoculation con-
ditions as the experiment reported here clearly demonstrated
that B. phytofirmans strain PsJN establishes endophytic popu-
lations. The presence of PsJN in the root and shoot interior
was reconfirmed in this study (data not shown).
Growth promotion. At 26°C, bacterized plantlets had six-
fold-higher total biomasses compared to those of the nonbac-
terized controls (Fig. 1). Although there was a significant (P ⬍
0.05) enhancement of the biomass of all organs, the stimula-
tion of root growth was the greatest: approximately 12-fold.
After 2 weeks of cold temperature treatment, the total biomass
decreased for both treatments, and a significant difference
between bacterized and control treatments was recorded for
only root biomass (Fig. 1).
Electrolyte leakage. Electrolyte leakage results show that
bacterization significantly (P ⬍ 0.05) improved the chilling
tolerance of the grapevine plantlets (Table 1). Prior to plantlet
exposure to the chilling treatment, ion leakage from leaves of
both bacterized and nonbacterized control plantlets was simi-
lar, 13 to 14% of the total leaf electrolytes. After chilling,
however, the electrolyte leakage was approximately twice as
great in the controls as in bacterized plantlets, 39 versus 20%,
respectively.
Free proline and total phenolics. Both cold and bacteriza-
tion treatments significantly (P ⬍ 0.005) increased free proline
content in leaves and stems in all tissues of the control plantlets
(Table 1). There was almost no proline found (it was either not
detected or detected in trace amounts) in the roots of the
control plantlets not subjected to chilling and 0.15 ␮mol · g⫺1
FW in the bacterized ones. Similarly, bacterization as well as
cold stress significantly (P ⬍ 0.05) enhanced the content of
total phenolics in leaves (Table 1). Independently of temper-
ature, bacterization enhanced the content of phenolics by ap-
proximately 50% and, independently of bacterization, low-
temperature treatment enhanced their titers by 10%.
Photosynthetic capacity. Independently of the temperature
treatment, bacterization significantly enhanced photosynthetic
activity of the grapevine leaves, compared to that of the control
(Table 2). When subjected to chilling, the photosynthetic ac-
tivities of both bacterized and nonbacterized plantlets de-
creased, although the cold treatment affected the nonbacte-
rized plantlets significantly (P ⬍ 0.05) more (Table 2). O2
evolution was also negatively affected by the low temperature
TABLE 2. Effect of chilling and bacterization on photosynthetic
activity (CO2 fixation and O2 production) and photosynthesis
of grapevine plantletsa
CO2 fixation O2 production Photosynthesis level
Treatment (␮mol m⫺2 (nmol min⫺1 (␮moles CO2 cm⫺2
s⫺1) cm⫺2) s⫺1)
Control at 26°C 3.15 a 0.30 a 0.47 a
Control at 4°C 1.51 b 0.15 b 0.15 b
Bacterized at 26°C 3.98 c 0.65 c 0.80 c
Bacterized at 4°C 2.68 d 0.33 d 0.40 a
a
Values correspond to the mean obtained with 24 plantlets for each replicate.
Three replicates were performed per treatment. In each row, the values repre-
sented by the same lowercase letters were not significantly different at a P value
of ⬍0.05 (Fisher’s protected LSD test). Photosynthetic activity is determined by
CO2 fixation and O2 production.
VOL. 72, 2006 B. PHYTOFIRMANS IMPROVES GRAPEVINE COLD TOLERANCE 7249

FIG. 2. Light micrographs of grapevine leaves. Twelve-week-old plantlets were subjected to 4°C treatment during 2 weeks, whereas the control
remained at 26°C. (a) Control plantlets. (b) Bacterized plantlets. (c) Control plantlets chilled at 4°C. (d) Bacterized plantlets chilled at 4°C. No
obvious correlation between cold treatment and marked host wall alterations or heavy tissue damages was observed. For both bacterized plantlets
grown at 26°C and bacterized chilled plantlets, the cross-section showed an accumulation of starch grains in leaf parenchyma as indicated by the
arrows. Ep, epidermis; P, parenchyma; S, starch. Magnification for panels a, b, and d, ⫻20; magnification for panel c, ⫻40.

treatment. In both temperatures, O2 evolution and CO2 fixa-
tion were significantly (P ⬍ 0.05) greater in the bacterized
plantlets.
Starch content and distribution. Compared to the nonbac-
terized controls, the enhanced photosynthetic activity of the
bacterized plantlets corresponded with significantly (P ⬍ 0.05)
higher starch content, particularly in leaves and shoots (Table
1). Chilling treatment had no significant (P ⬍ 0.05) effect on
starch content in all organs of the control plantlets (Table 1).
Under similar conditions, the bacterized plantlets continued
accumulating starch in leaves and shoots (P ⬍ 0.05). However,
no significant differences (P ⬍ 0.05) were found in roots be-
tween all treatments.
Microscopic observations. The vascular system is organized
into “bundles,” with water-conducting xylem vessels located
internally (on the pith side) and the food/organic material-
transporting phloem tissue on the exterior (see Fig. 3). No
obvious differences were observed between the temperature
treatments in the structure of the cell wall or the tissue orga-
nization in shoots and leaves of the nonbacterized plantlets
(Fig. 2 and 3). The nonbacterized control plantlets chilled at
4°C exhibited less growth but had larger vascular systems than
did the controls grown at 26°C (Fig. 4a and c). The chilling
treatment also caused cell wall alterations and some damage in
the epidermis and the cortex of control chilled roots (Fig. 4c).
The bacterized plantlets had secondary vascular structures,
with relatively large-diameter xylem cells, indicating enhanced
vascular cambial activity (Fig. 3b and d). The thick cell walls of
the xylem provide the principal structural support for aerial
plant parts. Microscopic examination of sections taken from
different organs confirmed the results of the starch analysis
reported in Table 1. More starch was observed in leaf and stem
sections of bacterized plantlets than in controls, with no clear
difference detected in roots (Fig. 4). Increased starch accumu-
lation in leaves of bacterized plantlets upon chilling could also
be seen on cross sections of leaf parenchyma (Fig. 2) and stele
(Fig. 3).
DISCUSSION
Burkholderia phytofirmans strain PsJN is capable of coloni-
zation of internal tissue while cocultured in vitro with grape
(12), tomato (34), and other plants (26). Based on the coloni-
zation pathway we observed with a PsJN::gfp2X derivative
tagged with green fluorescent protein (12) and on the effec-
tiveness of our surface sterilization protocol, which was proven
with grape (5, 12), tomato (34), and potato (17) plants, the
bacterium can be considered a true endophyte. The grape-
vine plantlets cocultured with PsJN grew faster and had
significantly (P ⬍ 0.05) more secondary roots (4, 5, 12). The
bacterium also enhances the resistance of tissue culture
plantlets to fungal disease (4, 5, 34) and heat stress (9). The
strain expresses a high level of 1-aminocyclopropane-1-car-
boxylate deaminase (33), the enzyme that hydrolyzes the
ethylene precursor 1-aminocyclopropane-1-carboxylate to
ammonia and ␣-ketobutyrate. By lowering the production of
this hormone in planta, the bacterium can decrease inhibi-
tory effects of ethylene on root elongation and its stimula-
tion of senescence under stress (19).
Despite the ability of plants to adapt partially to low-tem-
perature stress in temperate climates (30), plant growth and
overall productivity generally decline under chilling conditions
7250 AIT BARKA ET AL. APPL. ENVIRON. MICROBIOL.

FIG. 3. Light micrographs of grapevine shoots. Twelve-week-old plantlets were subjected to 4°C treatment during two weeks, whereas the
control remained at 26°C. (a) Control plantlets. (b) Bacterized plantlets. (c) Control plantlets chilled at 4°C. (d) Bacterized plantlets chilled at 4°C.
For both bacterized plantlets grown at 26°C and bacterized chilled plantlets, a cross-section showed an accumulation of starch grains in shoot stele
as indicated by the arrows. Bacterized plantlet shoots indicate the presence of secondary structure as evidenced by a net development of xylem
(black arrowheads). No obvious correlation between cold treatment and marked host wall alterations or heavy tissue damage was observed. Ep,
epidermis; Co, cortex; P, pith; Vs, vascular stele. Magnification, ⫻40.

(20). The extent of a plant’s ability to withstand such stress is
determined by metabolic alterations (32). In this study, we
demonstrated for the first time that the plant growth-promot-
ing bacteria colonizing grape plantlets can significantly influ-
ence the plantlets’ resistance to chilling. Plantlet bacterization
with Burkholderia phytofirmans strain PsJN had a pronounced
effect on grapevine growth, development, and responses to low
temperatures, i.e., diminished rates of biomass reduction and
electrolyte leakage during chilling and stimulated post-
chilling recovery.
In our previous study with frost-treated organs of grape-
vines, electrolyte leakage was a good indicator of plant sensi-
tivity to cold (1). The data thus indicate that the strain PsJN
may have the potential to lower the sensitivity of this crop to
chilling injury.
Bacterization significantly (P ⬍ 0.05) elevated the level of
proline and phenolics and enhanced the rate of photosynthesis
and starch deposition. This, combined with an enlarged root
system and improved sucrose uptake from the medium, may
have contributed to the stimulation of growth, development,
and adaptation to stress (29). Decrease of photosynthesis in-
duced by exposure to low temperatures is a well-known re-
sponse of chilling-sensitive plants (6, 27).
Our results show that the differences in cold tolerance be-
tween the control and bacterized plantlets were reflected by
differences in their abilities to significantly accumulate (P ⬍
0.05) carbohydrates under cold stress conditions. This is in
agreement with our earlier report of the accumulation of
starch in grapevine buds subjected to low temperatures (2).
This enhancement was explained in part by an inhibition of
amylase activity under cold conditions. In cabbage seedlings,
starch accumulated during cold acclimation and decreased
during deacclimation (31). A comparison between the changes
in chilling susceptibility of grapevine plantlets and their starch
content indicates that starch may also play a role in protecting
plant tissues against chilling.
Proline is a dominant organic molecule that accumulates in
many organisms upon exposure to environmental stress (14)
and plays multiple roles in plant adaptation to stress (24, 37),
including chilling (37, 40). We found a significant correlation
between freezing tolerance and an increase of proline concen-
tration in shoot and bud tissue of grapevines after exposure to
low temperatures (3). In this study, B. phytofirmans strain PsJN
significantly (P ⬍ 0.05) increased proline accumulation in
grapevine plantlets upon chilling. The enhancement of proline
accumulation in leaf tissues was also reported with an avirulent
strain of Pseudomonas syringae pv. tomato but not with the
isogenic virulent bacteria (16).
The observed accumulation of phenolic compounds induced
by PsJN confirms our earlier results (12). This phenomenon is
linked to the host defense response, which also includes the
strengthening of cell walls in the exodermis and several cortical
VOL. 72, 2006 B. PHYTOFIRMANS IMPROVES GRAPEVINE COLD TOLERANCE 7251

FIG. 4. Light micrographs of grapevine roots. Twelve-week-old plantlets were subjected to 4°C treatment during 2 weeks, whereas the control
remained at 26°C. (a) Control plantlets. (b) Bacterized plantlets. (c) Control plantlets chilled at 4°C. (d) Bacterized plantlets chilled at 4°C. Roots
of chilled plantlets showed obvious correlations between cold treatment and marked host wall alterations and tissue damage in the epidermis (Ep)
and the cortex (Co), as indicated by the arrows. Magnification, ⫻40.

cell layers. The activation of secondary responses associated
with the onset of induced resistance, including the oxidation
and polymerization of preexisting phenols and the synthesis of
new phenolic compounds via the activation of the phenylpro-
panoid pathway, has been demonstrated with another endo-
phytic bacterium, Serratia plymuthica, in cucumbers (8) and
with two PGPR strains (Pseudomonas fluorescens strain Pf4
and P. aeruginosa strain Pag) in chickpeas (35).
We reported earlier that B. phytofirmans strain PsJN pro-
tected grapevines against Botrytis cinerea (4, 5). The mecha-
nism of this protection was not localized but systemic. This
type of response usually confers an enhancement of plant re-
sistance to both biotic and abiotic stress (26). This phenome-
non is known as rhizobacteria-mediated induced systemic re-
sistance and was described as the mode of action of disease
suppression by nonpathogenic rhizosphere bacteria (39). Host
defense response pathways are preinduced by the colonizing
beneficial bacteria, allowing a much faster response to patho-
gen infection, i.e., formation of structural barriers, such as
thickened cell wall papillae due to the deposition of callose and
the accumulation of phenolic compounds at the site of patho-
gen attack (8, 12). Our results indicate that a plant’s reaction to
cold stress could be similar to the one previously reported
during pathogen attacks. Thus, the present results linked the
biotic stress to the abiotic stress in the way by which plants
react to the stress.
Conclusions. The concept of PGPR is now well established
(13), and some consideration of the relationship of PGPR to
biocontrol is worthwhile. Positive interactions between bacte-
rial endophytes and their host plants can result in a range of
beneficial effects, which are similar if not complementary to
those reported for exorhizobacteria. These include increased
plant growth and development, resistance to disease, and im-
provements in the host plant’s ability to withstand environmen-
tal stress (e.g., chilling).
B. phytofirmans strain PsJN has been well characterized as a
PGPR that triggers induced systemic resistance against fungal
pathogens (12, 34). Our findings indicate that this mechanism
also enhances grapevine resistance to cold stress.
The beneficial effect of endophyte bacteria may be through
their induction of the synthesis of proteins, which reduces the
development of symptoms, and also through the prevention of
some sets of reactions, which produce the symptoms of chilling
injury. Because the nucleation temperature of plants increases
with increasing population sizes of Ice⫹ bacteria, preemptive
competitive exclusion of Ice⫹ bacteria with naturally occurring
non-ice nucleation-active bacteria could be an effective and
practical means of frost control. The management of frost
injury by reducing Ice⫹ bacterial populations might become an
important new method of frost control.
Therefore, understanding molecular mechanisms behind the
chill-induced effects on photosynthesis by examining the myr-
iad changes in gene expression will be our next step toward a
better understanding of the role of PGPR in low-temperature
acclimation.
7252 AIT BARKA ET AL.
ACKNOWLEDGMENT
We thank Richard Veilleux, Department of Horticulture, Virginia
Polytechnic Institute and State University, Blacksburg, VA, for his
editorial advice.
REFERENCES
1. Ait Barka, E., and J. C. Audran. 1996. Utilisation de la conductivité
spécifique comme critère d’estimation de la viabilité au niveau de l’appareil
aérien des vignes champenoises soumises aux températures négatives. Can.
J. Bot. 74:413–418.
2. Ait Barka, E., and J. C. Audran. 1996. Réponse des vignes champenoises aux
températures négatives: Effet d’un refroidissement contrôlé sur les réserves
glucidiques du complexe gemmaire avant et au cours du débourrement. Can.
J. Bot. 74:492–505.
3. Ait Barka, E., and J. C. Audran. 1997. Response of champenoise grapevine
to low temperatures: Changes of shoot and bud proline concentrations in
response to low temperatures and correlations with freezing tolerance. J.
Hortic. Sci. Biotechnol. 72:577–582.
4. Ait Barka, E., A. Belarbi, C. Hachet, J. Nowak, and J. C. Audran. 2000.
Enhancement of in vitro growth and resistance to gray mold of Vitis vinifera
co-cultured with plant growth-promoting rhizobacteria FEMS Microbiol.
Lett. 186:91–95.
5. Ait Barka, E., S. Gognies, J. Nowak, J. C. Audran, and A. Belarbi. 2002.
Inhibitory effect of endophyte bacteria on Botrytis cinerea and its influence to
promote the grapevine growth. Biol. Control 24:135–142.
6. Antikainen, M., and M. Griffith. 1997. Antifreeze protein accumulation in
freezing-tolerant cereals. Physiol. Plant. 99:423–432.
7. Benhamou, N. 1996. Elicitor-induced plant defence pathways. Trends Plant
Sci. 1:233–240.
8. Benhamou, N., S. Gagné, D. L. Quéré, and L. Dehbi. 2000. Bacterial-medi-
ated induced resistance in cucumber: beneficial effect of the endophytic
bacterium Serratia plymuthica on the protection against infection by Pythium
ultimum. Phytopathology 90:45–56.
9. Bensalim, S., J. Nowak, and S. Asiedu. 1998. A plant growth promoting
rhizobacterium and temperature effects on performance of 18 clones of
potato. Am. Potato J. 75:145–152.
10. Bloemberg, G. V., and B. J. J. Lugtenberg. 2001. Molecular basis of plant
growth promotion and biocontrol by rhizobacteria. Curr. Opin. Plant Biol.
4:343–350.
11. Brüggemann, W., V. Beyel, M. Brodka, H. Poth, M. Weil, and J. Stockhaus.
1999. Antioxidants and antioxidative enzymes in wild-type and transgenic
Lycopersicon genotypes of different chilling tolerance. Plant Sci. 140:145–
154.
12. Compant, S., B. Reiter, A. Sessitsch, J. Nowak, C. Clément, and E. Ait
Barka. 2005. Endophytic colonization of Vitis vinifera L. by plant growth-
promoting bacterium Burkholderia sp. strain PsJN. Appl. Environ. Microbiol.
71:1685–1693.
13. Compant, S., B. Duffy, J. Nowak, C. Clément, and E. Ait Barka. 2005. Use of
plant growth-promoting bacteria for bioncontrol of plant diseases: principles,
mechanisms of action, and future prospects. Appl. Environ. Microbiol. 71:
4951–4959.
14. Delauney, A. J., and D. P. S. Verma. 1993. Proline biosynthesis and osmo-
regulation in plants. Plant J. 4:215–223.
15. Dobbelaere, S., J. Vanderleyden, and Y. Okon. 2003. Plant growth-promoting
effects of diazotrophs in the rhizosphere. Crit. Rev. Plant Sci. 22:107–149.
16. Fabro, G., I. Kovács, V. Pavet, L. Szabados, and M. E. Alvarez. 2004. Proline
accumulation and AtP5CS2 gene activation are induced by plant-pathogen
incompatible interactions in Arabidopsis. Mol. Plant-Microbe Interact. 17:
343–350.
17. Frommel, M. I., J. Nowak, and G. Lazarovits. 1991. Growth enhancement
and developmental modifications of in vitro grown potato (Solanum tubero-
sum ssp. tuberosum) as affected by a nonfluorescent Pseudomonas sp. Plant
Physiol. 96:928–936.
18. Gognies, S., A. Belarbi, and E. Ait Barka. 2001. Saccharomyces cerevisiae, a
potential pathogen towards grapevine, Vitis vinifera. FEMS Microbiol. Ecol.
1271:143–150.
19. Grichko, V. P. S., and B. R. Glick. 2001. Amelioration of flooding stress by
APPL. ENVIRON. MICROBIOL.
ACC deaminase-containing plant growth-promoting bacteria. Plant Phys.
Biochem. 39:11–17.
20. Haldiman, P. 1998. Low growth temperature induced changes to pigment
composition and photosynthesis in Zea mays genotypes differing in chilling
sensitivity. Plant Cell Environ. 21:200–208.
21. Lindow, S. E., and J. H. J. Leveau. 2002. Phyllosphere microbiology. Curr.
Opin. Biotechnol. 13:238–243.
22. Lodewyckx, C., J. Vangronsveld, F. Porteous, E. R. B. Moore, S. Taghavi, M.
Mezgeay, and D. van der Lelie. 2002. Endophytic bacteria and their potential
applications. Crit. Rev. Plant Sci. 21:583–606.
23. Martin, C., R. Vernoy, M. Carr, G. Vesselle, A. Collas, and C. Bougerey.
1987. The vine and techniques of in vitro cultivation. Bull. Org. Int. Vigne.
675:676:447–458.
24. Nanjo, T., T. M. Kobayashi, Y. Yoshida, Y. Kakubari, K. Yamaguchi-
Shinozaki, and K. Shinozaki. 1999. Antisense suppression of proline degra-
dation improves tolerance to freezing and salinity in Arabidopsis thaliana.
FEBS Lett. 461:205–210.
25. Nowak, J., and G. Lazarovits. 1997. Rhizobacteria for improvement of plant
growth and establishment. Hortic. Sci. 32:188–192.
26. Nowak, J., and V. Shulaev. 2003. Priming for transplant stress resistance in
in vitro propagation. In Vitro Cell. Dev. Biol. Plant 39:107–124.
27. Oquist, G., and N. P. Huner. 2003. Photosynthesis of overwintering ever-
green plants. Annu. Rev. Plant Biol. 54:329–355.
28. Ristic, Z., and E. N. Ashworth. 1993. Changes in leaf ultrastructure and
carbohydrates in Arabidopsis thaliana L. (Heynh) cv. Columbia during rapid
cold acclimation. Protoplasma 172:111–123.
29. Saladin, G., C. Clément, and C. Magné. 2003. Stress effects of flumioxazin
herbicide on grapevine (Vitis vinifera L.) grown in vitro. Plant Cell Rep.
21:1221–1227.
30. Saltveit, M. E. 2000. Discovery of chilling injury, p. 423–448. In S. D. Kung
and S. F. Yang (ed.), Discoveries in plant biology, vol. 3. World Scientific
Publishing, Singapore.
31. Sasaki, H., K. Ichimura, and M. Oda. 1996. Changes in sugar content during
cold acclimation and deacclimation of cabbage seedlings. Ann. Bot. 78:365–
369.
32. Seki, M., A. Kamei, K. Yamaguchi-Shinozaki, and K. Shinozaki,. 2003.
Molecular responses to drought, salinity and frost: common and different
paths for plant protection. Curr. Opin. Biotechnol. 14:194–199.
33. Sessitsch, A., T. Coenye, A. V. Sturz, P. Vandamme, E. Ait Barka, J. F. Salles,
J. D. van Elsas, D. Faure, B. Reiter, B. R. Glick, G. Wang-Pruski, and J.
Nowak. 2005. Burkholderia phytofirmans sp. nov., a novel plant associated
bacterium with plant beneficial properties. Int. J. Syst. Evol. Microbiol.
55:1187–1192.
34. Sharma, V. K., and J. Nowak. 1998. Enhancement of Verticillium wilt resis-
tance in tomato transplants by in vitro co-culture of seedlings with a plant
growth promoting rhizobacterium (Pseudomonas sp. strain PsJN). Can. J.
Microbiol. 44:528–536.
35. Singh, U. P., K. Birinchi, K. Sarma, and D. P. Singh. 2003. Effect of plant
growth-promoting Rhizobacteria and culture filtrate of Sclerotium rolfsii on
phenolic and salicylic acid contents in chickpea (Cicer arietinum). Curr.
Microbiol. 46:131–140.
36. Singleton, V. L., and J. A. Rossi. 1965. Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16:144–
158.
37. Sung, D. Y., F. Kaplan, K. J. Lee, and C. L. Guy. 2003. Acquired tolerance
to temperature extremes. Trends Plant Sci. 8:179–187.
38. Thomashow, M. F. 1999. Plant cold acclimation: freezing tolerance genes
and regulatory mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol.
50:571–599.
39. van Loon, L. C., P. A. H. M. Bakker, and C. M. J. Pieterse. 1998. Systemic
resistance induced by rhizosphere bacteria. Annu. Rev. Phytopathol. 36:453–
483.
40. Verma, D. P. S. 1999. Osmotic stress tolerance in plants: role of proline and
sulfur metabolism, p. 153–168. In K. Shinozaki and K. Yamaguchi-Shinozaki
(ed.), Molecular responses to cold, drought, heat and salt stress in higher
plants. R.G. Landes Company, Austin, Tex.
41. Wanner, L. A., and O. Junttila. 1999. Cold-induced freezing tolerance in
Arabidopsis. Plant Physiol. 120:391–400.