Plant Physiology and Biochemistry 42 (2004) 565–572

www.elsevier.com/locate/plaphy

Original article

Plant growth-promoting bacteria confer resistance

in tomato plants to salt stress
Shimon Mayak a,*, Tsipora Tirosh a, Bernard R. Glick b
a
Faculty of Agricultural, Food and Environmental Quality Sciences, The Robert H. Smith Institute of Plant Sciences & Genetics in Agriculture,
The Kennedy-Leigh Centre for Horticultural Research, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel
b
Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1
Received 1 March 2004; accepted 12 May 2004

Available online 15 June 2004

Abstract

The object of the work is to evaluate whether rhizobacteria populating dry salty environments can increase resistance in tomato to salt
stress. Seven strains of plant growth-promoting bacteria that have 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity were
isolated from soil samples taken from the Arava region of southern Israel. Following growth of these seedlings in the presence of 43 mM NaCl
for 7 weeks, the bacterium that promoted growth to the greatest extent was selected for further study. DNA analysis of the 16S RNA indicated
that the selected bacterium was Achromobacter piechaudii. This bacterium significantly increased the fresh and dry weights of tomato
seedlings grown in the presence of up to 172 mM NaCl salt. The bacterium reduced the production of ethylene by tomato seedlings, which was
otherwise stimulated when seedlings were challenged with increasing salt concentrations, but did not reduce the content of sodium. However,
it slightly increased the uptake of phosphorous and potassium, which may contribute in part to activation of processes involved in the
alleviation of the effect of salt. In the presence of salt the bacterium increased the water use efficiency (WUE). This may suggest that the
bacterium act to alleviate the salt suppression of photosynthesis. However, the detailed mechanism was not elucidated. The work described in
this report is a first step in the development of productive agricultural systems in saline environments.
© 2004 Elsevier SAS. All rights reserved.

Keywords: ACC deaminase; Lycopersicon esculentum; Plant growth-promoting bacteria; Salt stress

1. Introduction prove useful in developing strategies to facilitate plant

Natural soil forming processes in warm and dry regions
frequently produce saline soils with low agricultural poten-
tial. In these areas most crops are grown under irrigation, and
inadequate irrigation management leads to secondary salin-
ization that affects 20% of irrigated land worldwide. Soil
salinity is an enormous problem for agriculture under irriga-
tion. In addition to the use of traditional breeding and plant
genetic engineering with production of transgenic plants
[3,4,33], the use of plant growth-promoting bacteria may
Abbreviations: DW, dry weight; FW, fresh weight; FTW, fully turgid
weight; MS, Murashige & Skoog medium; OC, osmotic concentration;
PGPB, plant growth-promoting bacteria; RWC, relative water content;
WUE, Water use efficiency.
* Corresponding author.
E-mail address: mayaks@agri.huji.ac.il (S. Mayak).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.plaphy.2004.05.009
growth in saline soils.
Plant growth-promoting bacteria are free-living soil bac-
teria that can either directly or indirectly facilitate rooting
[22] and growth of plants [9]. Indirect stimulation of plant
growth includes a variety of mechanisms by which the bac-
teria prevent phytopathogens from inhibiting plant growth
and development [10,26,30]. Direct stimulation may include
providing plants with: fixed nitrogen, phytohormones, iron
that has been sequestered by bacterial siderophores, and
soluble phosphate. Many PGPRs also produce the enzyme
1-aminocyclopropane-1-carboxylate (ACC) deaminase and
metabolize ACC, a precursor to plant ethylene levels
[9,11,14,17], thus reducing the inhibition of root growth by
stress-induced ethylene.
Relatively few mechanisms have been unequivocally
demonstrated in explaining the increased resistance to envi-
ronmental stresses of plants treated with plant growth-
566 S. Mayak et al. / Plant Physiology and Biochemistry 42 (2004) 565–572

promoting bacteria. These include reduction of stress ethyl- Table 1

ene production via the action of ACC deaminase [13,15] and The effect of A. piechaudii ARV8 on the growth of tomato seedlings cv. F
144 irrigated with salt solution. Seven weeks after transplanting the see-
increased expression of the ERD15 gene that was previously dlings to pots, the fresh and dry weights were determined. Values are means
shown to be responsive to drought stress [19]. Nevertheless, of eight replicates ± S.E. Values with the same letter within columns indicate
plant growth-promoting bacteria found in association with no significant difference with P ≥ 0.05. The experiment was repeated six
plants grown under chronically stressful conditions, includ- times with similar results
ing high salinity, may have adapted to the stress conditions, Treatment Fresh weight (g) Dry weight (g)
and could provide a significant benefit to the plants. PGPR Salt concentration
In the present study, bacteria were isolated from soil (mM)
– – 6.60 ± 0.22 b 0.86 ± 0.05 b
samples collected in dry riverbeds in the Arava region in the
ARV8 – 7.57 ± 0.42 a 0.99 ± 0.06 a
southern part of Israel. Because of the arid conditions, the
– 43 5.99 ± 0.40 b 0.83 ± 0.05 b
soil is relatively salty. These samples are from sites that have ARV8 43 7.74 ± 0.33 a 1.12 ± 0.06 a
not been cultivated for hundreds of years, and thus biotic – 86 3.86 ± 0.06 d 0.57 ± 0.01 c
elements should be at a steady state within the soil. Bacterial ARV8 86 6.27 ± 0.25 b 0.99 ± 0.04 a
strains were selected to contain ACC deaminase [12] and – 172 2.86 ± 0.14 e 0.46 ± 0.03 c
should also act as plant growth promoters. Since these bacte- ARV8 172 4.28 ± 0.23 c 0.74 ± 0.05 b
ria are capable of lowering ethylene production in their host
plants, they should also render the plants more tolerant to accumulated fresh and dry weights to approximately the
salt-induced stress. same extent as non-inoculated plants grown in the absence of
salt.
As expected, salt affected the growth of the bacterium
2. Results (Fig. 2). In minimal medium, increasing salt concentrations
significantly delayed the onset of the exponential growth
2.1. Isolation of the best bacterial strain phase and slowed the rate of growth compared with rich
medium where the salt had only a small effect on bacterial
Seven bacterial strains capable of utilizing ACC as a sole growth.
source of nitrogen were isolated from soil samples from the
Arava region of Israel and used to inoculate the roots of 2.3. Salt influence root/shoot development
1-week-old tomato seedlings. The seedlings were then irri-
Monitoring the response of tomato plants to increasing
gated every other day with a 43 mM NaCl solution. At the
salt concentrations in the presence and absence of the bacte-
end of 7 weeks, the fresh and dry weights of the seedlings
rium A. piechaudii ARV8 in gnotobiotic growth pouches
were measured. Each of the seven bacterial strains promoted
permits an assessment of early plant responses to salt and a
the growth of the plants to some extent. The bacterium that
dissection of the effects on the roots as compared to the
promoted the largest increase in the weight of the plants was
shoots. The data presented in Table 2 demonstrate that the
selected for additional experiments. DNA sequence analysis
early plant response to salt, that is reduced growth, was
of its 16S RNA indicated that the selected strain was 99.9%
evident in shoots only at the higher concentration of 207 mM.
identical to Achromobacter piechaudii. The strain that was
However, with salt at concentrations of 120 and 207 mM the
selected for further study was therefore designated
A. piechaudii ARV8.

2.2. Resistance to salt stress

Inoculating tomato plants with A. piechaudii ARV8 and

then irrigating the plants with a range of salt concentrations
allowed for an evaluation of the effect of the bacterium on the
tolerance of the plants to salt. The dry and fresh weights
attained by the plants are given in Table 1. Plants inoculated
with A. piechaudii ARV8 grew to a significantly greater
extent than plants that were not treated with this bacterium.
The greater accumulation of fresh and dry weights by bacte-
rially treated plants occurred in both the absence and pres-
ence of salt. With increasing salt concentration, growth was
progressively inhibited in the absence of bacteria. To illus- Fig. 1. Effect of salt (NaCl 207 mM) on the growth of tomato seedlings. A
suspension of the bacterium A. piechaudii ARV8 was applied once to tomato
trate the relative inhibition, a photograph of the plants is seedlings during the second week after germination. Thereafter, the see-
presented in Fig. 1. Interestingly, plants inoculated with dlings were irrigated with salt (NaCl) solution. The picture was taken at the
A. piechaudii ARV8 and then irrigated with 86 mM salt age of 5 weeks.
 S. Mayak et al. / Plant Physiology and Biochemistry 42 (2004) 565–572 567

1.5

A
1

-1
E th ylen e n L .h .g
0

-1
0.5
7
12
O.D at 600 nm

2
B

1.5
0 12 Water NaCl ARV8 ARV8+NaCl

1
Fig. 3. Ethylene production by seedlings exposed to NaCl (207 mM) and to
0.5
7 the bacterium A. piechaudii ARV8. Five replicates were used for each
treatment. Vertical lines indicate S.E., n = 6. The experiment was repeated
0 four times with similar results.
0 5 10 15 20 25

Time (h)
results when plants are subjected to high salt concentrations.
Fig. 2. The effect of NaCl on the growth of A. piechaudii ARV8 in DF Here, as the salt concentration was increased, the RWC
minimal medium (A), YT rich medium (B). Numbers correspond to concen- decreased; this occurred to nearly the same extent in bacteri-
tration of NaCl in g l–1, n = 3. The experiment was repeated three times with
ally treated and untreated plants (Table 3). At the same time,
similar results.
raising the concentration of salt in the irrigation medium
root lengths are decreased. Inoculated seedlings responded from 0 to 120 mM resulted in an increase of the osmotic
differently to salt. At a salt concentration of 120 mM, shoot concentration (OC) of the cell sap from 292.8 to 462.3 mmol
and root elongation was slightly stimulated. Inoculated kg–1 to (Table 3), while irrigating with salt solution 207 mM
shoots treated with 120 mM salt grew to 32.8 mm while did not cause any further increase in the osmotic concentra-
inoculated shoots irrigated with water grew to 27.4 mm. At tion.
the same time, the higher concentration of salt (207 mM)
suppressed elongation of shoots of non-inoculated (compare 2.6. Bacterial strain influence WUE
17.8 mm with 26.0 mm) and inoculated seedlings (compare
22.6 mm with 27.4). Similar results were obtained with roots. WUE refers to the ratio between the accumulated biomass
2.4. Monitoring ethylene production to transpiration or water use. The response of the stomata is
assumed to be a major component influencing water loss and
A rise in ethylene production occurred in seedlings chal- at the same time it also regulates the supply of carbon to
lenged with NaCl (Fig. 3). At the same time, the production photosynthesis. Therefore, it was of interest to evaluate how
of ethylene by seedlings not challenged with salt remained this parameter was affected by the bacterium, which the data
low. When seedlings were treated with a suspension of indicated was capable of increasing the resistance of plants to
A. piechaudii ARV8 and then challenged with NaCl, ethyl- salt stress.
ene production was significantly lower than in the absence of On the basis of water used by the plant, the bacterium has
the bacterium. increased the efficiency of tomato seedlings in the production
of biomass. This was reflected in both WUE calculations: one
2.5. Salt induce stress in plants that is based on accumulated fresh weight and the second that
Relative water content (RWC) is a useful measure of the is based on dry weight accumulation as well (Table 4).
physiological water status of plants [8]. Water stress often Irrigating with salty water reduced both biomass production
Table 2 Table 3
The effect if A. piechaudii ARV8 on the elongation of roots and shoots of The effect of A. piechaudii ARV8 on the relative water content (RWC) and
tomato seedlings cv. F 144 in gnotobiotic growth pouches, irrigated with salt on the osmotic concentrations (OC), in shoots of tomato seedlings cv. F
solution. Values with the same letter within columns indicate no significant 144 grown in small pots and irrigated with salt solution. Values with the
difference with P ≥ 0.05. Values are means ± S.E., n = 4 replicate pouches same letter within columns indicate no significant difference with P ≥ 0.05.
each containing 10 seedlings. The experiment was repeated four times with Values are means ± S.E., n = 6. The experiment was repeated three times
similar results with similar results
PGPR Salt concentration Shoots (mm) Roots (mm) PGPR Salt concentration RWC OC (mmol kg–1)
(mM) (mM)
– 0 26.0 ± 0.9 b,c 77.1 ± 5.2 b – 0 0.872 ± 0.010 a 292.8 ± 6.5 c
– 120 27.2 ± 1.6 b 69.4 ± 6.0 c – 120 0.774 ± 0.005 b 462.3 ± 11.6 a
– 207 17.8 ± 2.2 d 45.0 ± 5.9 d – 207 0.630 ± 0.022 d 369.5 ± 31.4 b
ARV8 0 27.4 ± 1.2 b 77.1 ± 3.9 b ARV8 0 0.879 ± 0.008 a 280.5 ± 4.0 c
ARV8 120 32.8 ± 1.8 a 82.3 ± 3.2 a ARV8 120 0.732 ± 0.029 c 430.8 ± 18.9 a
ARV8 207 22.6 ± 0.5 c 53.0 ± 6.1 d ARV8 207 0.639 ± 0.012 d 413.2 ± 26.6 a
568 S. Mayak et al. / Plant Physiology and Biochemistry 42 (2004) 565–572

Table 4
The effect of A. piechaudii ARV8 on WUE of tomato seedlings cv. F144 grown in specially designed growth units and irrigated with salt solution as described
in Section 4. Plants were inoculated with A600 nm = 1.0 and irrigated with NaCl (172 mM). Values with the same letter within columns indicate no significant
difference with P ≥ 0.05. Values are means ± S.E., n = 10. The experiment was repeated three times with similar results
Treatment WUE (DW) WUE (FW) DW (mg) FW (g)
(mg biomass Mg–1 H2O)
Water 2.30 ± 0.06 c 19.37 ± 0.32 c 520.8 ± 16.8 4.39 ± 0.13
ARV8 3.31 ± 0.22 a 30.36 ± 2.05 a 885.2 ± 36.9 8.07 ± 0.23
NaCl 2.72 ± 0.14 b 19.76 ± 0.97 c 840.0 ± 21.8 3.94 ± 0.16
ARV8 + NaCl 2.90 ± 0.12 a,b 23.14 ± 0.83 b 794.9 ± 38.9 6.33 ± 0.25

and the WUE. Evaluating the combined effect of salt and the
bacterium indicate that the bacterium restrains the reduction
in WUE that is otherwise imposed by salt.
2.7. Bacterial strain influence ion accumulation
Ion accumulation in the shoots of tomato plants was af-
fected by irrigation with salt (Table 5). The content of Ca,
Mg, K and S was reduced by the salt treatment; the content of
Na and P was increased; and the content of Ba and Fe was
unchanged. The main effect of the presence of the bacterium
was an increase in the concentration of K in the presence of
salt.
3. Discussion
Saline conditions are known to suppress the growth of
plants [5]. With increasing concentrations of salt in the irri-
gation solution, greater reductions of growth are observed
(Table 1). However, when plants were treated with a suspen-
sion of A. piechaudii ARV8, the extent of growth suppression
was decreased and the bacterially treated plants accumulated
more fresh and dry weights than untreated plants (Table 1).
This clearly demonstrates that the selected bacterium can
alleviate some of the debilitating effects of salt stress.
Measurements of the elongation of shoots and roots of
small seedlings indicate that roots compared with the shoots
responded to lower concentrations of salt, resulting in greater
suppression of elongation (Table 2). This may be due to the
fact that the roots are in intimate contact with the salt solution
while the shoots experienced presumably lower salt concen-
tration, i.e. only that which was taken up by the seedlings.
Increased production of ethylene in response to salt stress
has been previously demonstrated in tomato [26] and cucum-
ber plants [7]. Based on similar results Abeles [1] has coined
Table 5
Ion concentration in the shoots of salt treated tomato plants inoculated and non-inoculated with A. piechaudii ARV8. Values are ×103 mg kg–1. Values are means
of five replicates ± S.E. Values with the same letter within columns indicated no significant difference with P ≥ 0.05. The data is of an experiment that was
repeated twice with similar results
Treatment Ca Mg K Na
Control 26.60 ± 1.16 a 32.2 ± 1.1 a 28.7 ± 1.4 a
NaCI 7 g l–1 11.40 ± 0.66 26.6 ± 0.8 b 16.5 ± 0.7 c
ARV8 + NaCI 7 g l–1 9.16 ± 0.36 c 21.6 ± 1.8 c,d 21.5 ± 1.8 b
NaCI 12 g l–1 12.46 ± 1.07 b 25.5 ± 1.6 b 21.0 ± 1.9 b
ARV8 + NaCI 12 g l–1 9.67 ± 0.32 c 23.4 ± 1.0 c,b 24.6 ± 1.6 a,b
the term “stress ethylene”. Increased salt concentration de-
creases the osmotic potential of a growth medium, thus
reducing the water availability. The mechanism of response
to salt stress is thought to be in part similar to the mechanism
of response to drought stress [4]. A clear rise in ethylene
production was measured in response to desiccation of de-
tached leaves [2]. On the other hand, when Morgan and Drew
[24] observed that a rise in ethylene occurred in response to
rapid drying, but slow drying did not promote ethylene pro-
duction, this result was interpreted to indicate that the stimu-
lated ethylene production is a wound response [24]. At vari-
ance with this conclusion, Mayak [21] observed that a rise in
ethylene production in cut carnation flowers occurred during
the recovery period that followed transient desiccation; an
observation at odds with the notion that ethylene is strictly a
response to wound. The literature collectively indicates that
in as much as ethylene is involved in a plant’s response to
abiotic stresses, plants vary in their response. Some plants
respond to stress with a rise in ethylene production, while in
other plants ethylene production remains unchanged. Sensi-
tivity to ethylene may also play a role in the response to stress
[20,34], which means that ethylene may participate in the
regulation of plant’s response to stress without noticeable
changes in its concentration.
The present study demonstrates that ethylene increased
concurrent with salt stress in tomato seedlings. Ethylene
influences various phases of vegetative growth in plants re-
sulting in overall reduced growth [31]. Furthermore, ethylene
is involved in transduction of a signal between the recogni-
tion of salt stress and the response in terms of physiological
processes [36].
In many instances, removing or blocking the effect of
stress ethylene results in alleviation of the stress effect. Since
the selection procedure [9] employed in the present study for
isolation of A. piechaudii ARV8 favors the growth of bacteria
containing ACC deaminase, inoculating stressed plants with
P S Ba Fe
1.8 ± 0.2 c 0.8 ± 0.04 c 5.4 ± 0.10 a 0.23 ± 0.01 a 0.11 ± 0.01 a
16.6 ± 1.3 b 1.0 ± 0.07 b 3.1 ± 0.04 b 0.20 ± 0.01 a 0.11 ± 0.01 a
12.7 ± 1.3 b 1.7 ± 0.18 a 3.6 ± 0.15 b 0.19 ± 0.02 b 0.09 ± 0.01 b
33.5 ± 3.4 a 1.1 ± 0.08 b 3.3 ± 0.10 b 0.17 ± 0.02 c 0.08 ± 0.01 b
35.5 ± 2.6 a 1.7 ± 0.09 a 3.3 ± 0.25 b 0.18 ± 0.01 c 0.09 ± 0.01 a,b
 S. Mayak et al. / Plant Physiology and Biochemistry 42 (2004) 565–572
these bacteria should reduce the ethylene concentration (a
consequence of reduction in the level of ACC, the immediate
precursor of ethylene) [e.g. 16]. Furthermore, in Glick’s
laboratory a dozen bacterial strains were isolated and in all of
them that are capable of utilizing ACC as sole source nitro-
gen, they were also found to contain ACC deaminase. In
addition, when the ACC deaminase activity of ARV8 was
measured, it was found to be in the range that was previously
reported for other plant growth-promoting bacteria [29]. The
results presented in Fig. 3 supports this expectation, namely
reduced ethylene production following inoculation has in-
deed occurred. Furthermore, we suggest that lower ethylene
alleviates the effect of stress.
In addition to a stress response mechanism involving
reduction in ethylene production, other mechanisms may
also play a role. In line with this proposal we have analyzed
the effect of the bacterium on changes in additional param-
eters, including RWC, OC and WUE.
In response to an increased salt concentration, a reduction
in tomato seedlings relative water content occurred. The use
of RWC is a good indicator of water stress [8] and reduced
RWC clearly demonstrates that the seedlings are experienc-
ing stress. However, since the level of RWC was similar in
the salt treated and non-treated plants (Table 3) we interpret
the results as demonstrating that the bacterial treatment did
not influence the extent of water stress. Nevertheless, the
bacterium improved the growth of the salt-treated plants.
Associated with the reduction in relative water content
influenced by irrigation with a salt solution, an increase in the
osmotic concentration has occurred (Table 3). The increase
in osmotic concentration in bacterially treated plants was
similar to the increase in untreated plants, suggesting that
neither reduction nor stimulation of mineral uptake mecha-
nisms is activated by the bacterium.
WUE is a measure of the capability of a plant to accumu-
late biomass per unit of water used by the plant. This param-
eter is valuable especially in a water limited environment.
One of the major changes occurring in response to reduction
in water availability is induction of closure of stomata, thus
restricting water loss. However, closure of stomata limits gas
exchange resulting in a decrease in photosynthesis [18].
Challenging seedlings with salt raised the uptake of salt by
the seedlings as indicated by a rise in OC (Table 3), resulting
in a water stress situation reflected by a reduction in RWC.
Because of the expected closure of stomata, a reduction in
gas exchange is likely to follow. Consequently, a rise in the
WUE ratio should also occur. Our data demonstrate that
treating seedlings with salt did not change the WUE
(Table 4), which in accordance with our previous assumption
should reflect a reduction in photosynthesis. The bacterium
raised the WUE, which is either due to increased photosyn-
thesis or a reduction in transpiration. A combined treatment
that includes salt and the presence of the bacterium slightly
reduced the WUE suppression observed when seedlings were
challenged with salt (Table 4), at least when calculating on
the basis of fresh weight. We interpret these results to indi-
569
cate the dominant effect was a consequence of the bacterium
enhancing photosynthesis. This conclusion is supported by
the gain in biomass (DW) amounting to a rise of +52.6%
compared to a rise of +3.6% (compare DW gain in ARV8 +
NaCl treatment with NaCl treatment in Table 4).
Although the experimental design used in measurements
of ion accumulation does not allow us to dissect the distribu-
tion of ions between the intracellular compartments, some
preliminary suggestions can be debated. The strategies for
maintenance of a high K+/Na+ ratio in the cytosol evolve
around regulating the movement of K+ to and from the
cytosol. Different K+ channels were identified relating to
sodium uptake [28,35]. Also, extrusion of Na+ by plasma
membrane H+-ATPase is active in the regulation of low
cytosolic Na+ concentration [32]. In addition, compartmen-
talization of Na+ into the vacuoles prevents toxic effects of
Na+ in the cytosol [4]. Collectively, the activity of the chan-
nels and pumps maintain Na+ at low, and K+ at high, concen-
tration. The results of the analysis of the content of mineral
elements (Table 5) indicate no statistical difference in the
concentration of sodium with and without the bacterium,
negating the possibility that the bacterium exerts its plant
growth-promoting effect via a mechanism that lowers the
content of sodium.
Interestingly, the bacterium influenced an increase in the
content of phosphorus (Table 5). Thus, A. piechaudii ARV8,
in addition to amelioration of salt stress may also act by an as
yet an unknown mechanism to enhance phosphorous solubi-
lization or uptake of phosphate.
In summary, the present study identified a bacterium con-
taining ACC deaminase that increases the resistance of to-
mato seedlings to salt. The bacterium reduces the production
of ethylene by the seedlings hence alleviating the suppres-
sion of growth. The bacterium did not influence the status of
water parameters. It did, however, increase the WUE. In
addition, an alternative explanation cannot be ruled out. That
is, the combined effect, the bacterium promoting effect and
the salt suppression effect may be independent of one other.
The data presented here suggest that, without any genetic
manipulation of the plant, it may be possible to productively
cultivate a variety of crop plants under saline conditions,
provided that the plants are grown in the presence of a
suitable plant growth-promoting bacterium such as
A. piechaudii ARV8.
4. Methods
4.1. Bacterial isolation
Bacterial strains were isolated according to [9] from
rhizosphere soil samples from Lycium shawii plants growing
in dry riverbeds in the Arava region (N + 31°10′ L + 35°22′)
in southern Israel where the annual rainfall is below 50 mm.
Bacterial suspensions for the treatment of plants were pre-
pared as described previously [23]. Essentially, seven bacte-
570 S. Mayak et al. / Plant Physiology and Biochemistry 42 (2004) 565–572
rial colonies from solid DF medium containing ACC as a sole
source of nitrogen were used to inoculate YT medium and
then incubated for 24 h with vigorous shaking (i.e. approxi-
mately 250 rpm) to ensure proper aeration. Following
growth, the bacterial cells were pelleted by centrifugation at
5000 × g for 10 min. The pellet was then re-suspended in
distilled water. This step was repeated twice before the bac-
terial suspension was adjusted to A600 nm = 1.0. Forty millili-
ters of each bacterial suspension were applied once to
2-week-old seedlings. The tomato seedlings were fertilized
and challenged with NaCl as described in the next section. At
7 weeks, based on general appearance and plant size, the
bacterium that induced the best growth was selected for
further study.
4.2. Plant material and growth conditions
Tomato (Lycopersicon esculentum Mill cv. F144) seeds
were sown in trays in wet vermiculite. After 1 week, uniform-
sized seedlings were selected and planted in vermiculite, one
per 7-cm plastic pot. During the second week, the seedlings
were fertilized once with 40 ml of 1/5 Murashige and Skoog
(MS) medium [25]. Three days after fertilization some of the
seedlings were treated with 40 ml of bacterial suspension
(A600 nm = 1.0), while others were watered with de-ionized
water. During the rest of the experiment the seedlings were
irrigated with a NaCl solution (0, 43 or 207 mM). The
seedlings were maintained in a growth chamber at a
day/night temperature of 25/20 °C with ~100 µmol photons
m–2 s–1 of light supplied for 12 h during the daytime.
4.3. Monitoring ethylene production by seedlings
in response to stress
Thirty seeds were imbibed in either a bacterial suspension
(A600 nm = 1.0) or deionized water for 2 h and then the
suspension or water was drained and the seeds were placed
inside 25 ml Erlenmeyer flasks on a piece of filter paper. Two
milliliters of de-ionized water were added to each flask. After
8 days, when the cotyledons were expanded, the excess
liquid was drained and 2 milliliters salt solution (207 mM
NaCl) was added. Seeds that were imbibed in de-ionized
water and were not treated with salt served as a control. Four
hours after the addition of salt, the flasks were closed for 2 h
with a rubber septum and the ethylene in the headspace was
sampled and measured by gas chromatography. Six flask
replicates were used for each treatment.
4.4. Water status and osmotic concentration
After about 8 weeks (or as indicated) plants were har-
vested and fresh (FW) and dry (DW) weights were measured.
For each plant, the fully turgid weight (FTW), defined as the
weight of the shoot after the plant was held at 100% humidity
at 4 °C for 48 h, was recorded. The RWC was calculated,
where: RWC = (FW – DW)/(FTW – DW). Six plant repli-
cates were used for each treatment.
The osmotic concentration of the cell sap that was ex-
tracted from shoot tissue was measured. Freezing and thaw-
ing the tissue twice facilitated extraction of the cell sap from
the tissue, then the osmotic concentration of the sap was
measured using a Wescor model 5100C vapor pressure os-
mometer (Wescor Inc., Logan, UT, USA). Six plant repli-
cates were used for each treatment.
4.5. Determination of WUE
Tomato (L. esculentum Mill cv. F144) seedlings were
started from seeds and treated and maintained as described in
section plant material and growth conditions. Two seedlings
were transferred to a specially designed growth unit. The
growth unit was constructed to allow for conveniently moni-
toring the added and transpired water by the plants. The
growth unit consisted of two polycarbonate culture boxes,
one stacked into the other to form a watertight seal (Fig. 4).
Through the bottom of the upper unit a 0.5 cm hole was
drilled to allow for drainage of excess solution. Following
irrigation, part of the solution applied was retained by the
vermiculite packed in the upper unit while the other part of
this solution was drained into the lower unit becoming un-
available to the plants. Following application of additional of
liquid (salt solution, 172 mM NaCl or deionized water) the
growth unit was weighed. A few days later the growth unit
was weighed again and the difference corresponded to tran-
spiration (an estimation of the water used by the plant during
this time period). Growth units containing properly wetted
vermiculite, but without plants were maintained along side
the treated growth units. The reduction in weight during
consecutive measurements corresponds to evaporation, and
this value was deducted from the transpiration raw data.
When the lower unit accumulated solution to capacity, it was
drained and the weight of the liquid was recorded, to account
for solution that was neither transpired nor evaporated. The
plants were fertilized as described above in the section on
plant material and growth conditions. They were irrigated
with water containing 172 mM NaCl. At the end of the
experiment (8 weeks), the plants were harvested and fresh
Upper unit
Drain hole in the
bottom of the Lower unit
upper unit
Growth unit
Fig. 4. A diagram of the growth unit used to monitor the WUE.
 S. Mayak et al. / Plant Physiology and Biochemistry 42 (2004) 565–572
and dry weights were determined. The accumulation of bio-
mass (fresh and dry weights) divided by the water transpired
reflects the WUE. Growth units irrigated with de-ionized
water served as a control. Ten growth unit replicates were
used for each treatment.
4.6. Solute analyses
Mineral elemental analysis of plant extracts was con-
ducted using inductively coupled plasma atomic emission
spectrometry with an ICP-AES model “Spectroflame”
(Spectro; Kleve, Germany).
4.7. Pouch assay
To measure the effect of added bacteria on early root and
shoot growth, seeds were imbibed in either double distilled
water or a suspension of the test bacterium for 3 h [27]. The
absorbance of the bacterial suspension was adjusted to
1.0 O.D. at 600 nm prior to incubation with the seeds. Ten
seeds with the same treatment were placed in each sterile
growth pouch (Northrup King Co., Minneapolis, MN, USA).
The pouches were placed upright in a rack covered with
Saran Wrap™ and kept at 22 ± 1 °C for 9 d, with a light level
of about 14 µmol m–2 s–1. Four growth unit replicates were
used for each treatment.
4.8. Bacterial growth curve
YT (Yeast extract-tryptone; Difco; Detroit, MI, USA) or
DF minimal medium [6], to which NaCl at various concen-
trations (0, 120 and 207 mM) was added, were inoculated
with the isolated strain A. piechaudii. The cultures were
incubated in a shaking water bath operated at 200 rpm and
maintained at 30 °C. A. piechaudii ARV8 Growth was moni-
tored as the change in optical density (OD) at 600 nm.
4.9. Statistical analysis
Data were analyzed by analysis of variance (ANOVA),
and pair wise comparisons were done using a Student’s t-test.
All hypotheses were tested at the 95% confidence level.
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