Plant Science 166 (2004) 525530

Plant growth-promoting bacteria that confer resistance to water

stress in tomatoes and peppers
Shimon Mayak a, , Tsipora Tirosh a , Bernard R. Glick b
a

The Kennedy-Leigh Centre for Horticultural Research, Faculty of Agriculture, Food and Environmental Quality Sciences,
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 26 September 2002; received in revised form 21 October 2003; accepted 27 October 2003

Abstract
The work reported here evaluates whether bacteria populating arid and salty environments can confer resistance in tomato and pepper
plants to water stress. Plant growth-promoting bacteria that have ACC deaminase activity were isolated from soil samples taken from the
Arava region of southern Israel. One of these strains, Achromobacter piechaudii ARV8 [Mayak et al., Plant growth-promoting bacteria that
confer resistance in tomato and pepper plants to salt stress, submitted for publication.] significantly increased the fresh and dry weights of
both tomato and pepper seedlings exposed to transient water stress. In addition, the bacterium reduced the production of ethylene by tomato
seedlings, following water stress. During water deprivation the bacterium did not influence the reduction in relative water content; however,
it significantly improved the recovery of plants when watering was resumed. Inoculation of tomato plants with the bacterium resulted in
continued plant growth during both the water stress and after watering was resumed. Based on the results of the experiments reported herein,
the use of plant growth-promoting bacteria such as A. piechaudii ARV8 may provide a means of facilitating plant growth in arid environments.
2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Tomato; Pepper; PGPB; Water stress; Drought; RWC

1. Introduction
Plant growth-promoting bacteria are free-living soil bacteria that can either directly or indirectly facilitate the
growth of plants [2,3]. Indirect stimulation of plant growth
includes a variety of mechanisms by which the bacteria prevent phytopathogens from inhibiting plant growth and development [4,5]. Direct stimulation may include providing
plants withfixed nitrogen, phytohormones, iron that has
been sequestered by bacterial siderophores, soluble phosphate, or the enzyme ACC deaminase that can lower plant
ethylene levels [68]. The suggested role of ACC deaminase is to ensure that the concentration of ethylene does
not become elevated to the point that growth (especially of
roots) is retarded [3,6,7]. Thus, mutant bacteria that lack
ACC deaminase activity are no longer able to lower plant
ethylene levels and thereby promote root elongation [911].

Coresponding author. Tel.: +972-8-9489335; fax: +972-8-9468263.

E-mail address: mayaks@agri.huji.ac.il (S. Mayak).

In addition to facilitating the growth of plant roots, plant

growth-promoting bacteria can, protect plants from the
deleterious effects of some environmental stresses [12,13]
including heavy metals [14], flooding [15], salt [1] and
phytopathogens [16].
Relatively few mechanisms have been unequivocally
demonstrated to explain the increased resistance to environmental stresses including water stress of plants treated
with plant growth-promoting bacteria. The mechanisms that
have been suggested include reduction of stress ethylene
production via the action of ACC deaminase [12] and increased expression of the ERD15 gene, which is responsive
to drought stress [13,17].
In the present study, we evaluated the effect of the bacterium Achromobacter piechaudii ARV8 on the performance
of tomato and pepper plants under drought stress conditions.
The bacterium was recently isolated from soil samples collected in dry riverbeds in the Arava region in the southern
part of Israel where rainfall is scarce. This bacterial strain
was selected to contain ACC deaminase [2] and thus should
be able to lower ethylene production in its host plants; and

0168-9452/$ see front matter 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2003.10.025

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S. Mayak et al. / Plant Science 166 (2004) 525530

according to a previously proposed model [3], it should also

render the plants more tolerant to drought stress. In fact, the
bacterium was found to be both capable of lowering ethylene production in its host plants, and of ameliorating some
of the effect of drought stress.

2. Materials and methods

2.1. Bacterial strain and growth
The bacterium Achromobacter piechaudii ARV8 was isolated as described by Glick et al. [9] from a rhizosphere soil
sample from a Lycium shawii plant growing in a dry riverbed
in the Arava region of southern Israel, reported previously
[1]. Bacterial suspensions for the treatment of plants were
prepared as described previously [18]. Essentially, a single
bacterial colony from solid DF medium containing ACC as
a sole source of nitrogen was used to inoculate YT medium
and then incubated for 24 h with vigorous shaking (i.e. approximately 250 rpm) to ensure proper aeration. Following
growth, the bacterial cells were pelleted by centrifugation
at 5000 g for 10 min and then re-suspended in distilled
water. This step was performed twice before the bacterial concentration was adjusted to 1.0 absorbance unit at
600 nm. The latter suspension was used to irrigate plants as
indicated.
2.2. Plant material and growth conditions
Tomato (Lycopersicum esculentum Mill cv. F144) and
pepper (Capsicum annuum L. cv. Maor) seedlings were
started from seeds that were sown in plastic trays in wet
vermiculite. After 1 week, uniform sized seedlings (shoot
height approximately 3 cm) were selected and planted in
vermiculite, one per 7 cm diameter plastic pot. During the
2nd week the seedlings were fertilized once with 40 ml
of either 1/10 or 1/5 Murashige and Skoog (MS) medium
[19] as indicated. 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. Two weeks after the seedlings were transplanted, watering ceased and only resumed after 7 or 12
days as indicated. The seedlings were maintained in a
growth chamber at a day/night temperature of 25/20 C
with 25 mol photons m2 s1 or 75 mol photons m2 s1
of light supplied for 12 h during the daytime.
2.3. Monitoring plant growth
Fresh (FW) and dry (DW) weights of tomato plants were
measured 5 weeks after germination. Similar experiments
were also conducted with pepper plants except that harvesting the plants and measurements were done 7 weeks after
germination. In other experiments, FW and DW of tomato
plants were also measured up to five times (0, 6, 12, 17 and

23 days) starting at 3 weeks after germination and continued

thereafter at times indicated.
2.4. Ethylene production by seedlings in response
to stress
Thirty seeds were placed in each 25 ml Erlenmeyer flask
on sterile filter paper before 2 ml of water were added. Seeds
were imbibed in water or a bacterial suspension (A600 nm =
1.0) for 2 h before being placed in the flasks. After 8 days,
when the cotyledons were expanded, the excess water was
removed. The flasks were closed for 2 h with a rubber septum and the ethylene in the headspace was sampled and measured by gas chromatography (Shimadzu model GC-17A).
At the end of the ethylene accumulation period the stoppers
were removed and the flasks were maintained in a growth
chamber as described above except that the light irradiance was 10 mol photons m2 s1 . Whereas water was not
added to the stressed seedlings for the rest of the experiment, sufficient water was added to each flask of the appropriate controls to be continuously visible on partially tilting
the flask.
2.5. Water status
At times 0, 6, 12, 17 and 23 days, starting at 3 weeks after germination the relative water content (RWC) in tomato
plants was determined. The fully turgid weight (FTW), defined as the weight of the shoot after the plant was held in
100% humidity conditions in the dark at 4 C for 48 h, of
each plant was recorded. The relative water content (RWC)
was calculated, where:
RWC =

FW DW
FTW DW

2.6. Statistical analysis

Data were analyzed by analysis of variance (ANOVA),
and pair wise comparisons were done using a students t-test.
All hypotheses were tested at the 95% confidence level.

3. Results
The bacterium A. piechaudii ARV8 was isolated from a
soil sample from a desolate region in the southern part of Israel, where the annual rainfall is below 50 mm and the land
has not been cultivated for many hundreds of years. The
isolated bacterium was used to inoculate 10-day-old tomato
seedlings and the dry and fresh weights of seedlings treated
with A. piechaudii ARV8 were compared with those obtained from seedlings treated with the well-established plant
growth promoting bacterium Pseudomonas putida GR12-2
[20,21]. Plants not treated with any bacterium served as an
additional control. In this experiment, both P. putida GR12-2

S. Mayak et al. / Plant Science 166 (2004) 525530

Table 1
The effect of ACC deaminase-containing plant growth-promoting bacteria
on the growth of tomato seedlings cv. F-144
Strain

Fresh weight (mg)

Dry weight (mg)

No bacterium added
P. putida GR12-2
A. piechaudii ARV8

155
412 22.4b
574 58.8a

15.60.65c
41.02.12b
59.75.43a

7.20c

During the 2nd week tomato seedlings grown in small pots were inoculated
with a bacterial suspension. By the end of the 4th week watering was
stopped for 2 weeks, and then watering was resumed. After 5 weeks fresh
and dry weights were determined. Values are means of 10 replicates
S.E. Superscripted letters indicate values within the same column that are
either statistically significantly different (when the letters are different) or
not (when the letters are the same).

and A. piechaudii ARV8 promoted plant growth to a significant extent compared to the non-bacterized control, with A.
piechaudii ARV8 promoting growth significantly more than
Strain p. putida GR12-2 (Table 1).

527

Table 2
The effect of ACC deaminase-containing plant growth-promoting bacterium A. piechaudii ARV8 on the growth of pepper seedlings cv. Maor,
exposed to transient water stress
Treatment

FW (mg)

DW (mg)

No bacterium added
A. piechaudii ARV8

560 50b
960 60a

70 0.16b
120 2.60a

Pepper seedlings were managed essentially as outlined in Table 1, except

for few changes. By the end of 7 weeks after seed germination the fresh
and dry weights were determined. Values are the means of 6 replicates
S.E. Values with the same superscripts within columns indicate no
significant difference with P 0.05.

To test whether the bacterium has the same effect on

other plants, it was used to treat pepper seedlings subjected
to the same regime as the tomato seedlings, and at the
end of 7 weeks their fresh and dry weights were recorded.
The data presented in Table 2 indicates that A. piechaudii

Fig. 1. The effect of the ACC deaminase-containing plant growth promoting bacterium A. piechaudii ARV8 on growth (fresh weight (FW) and dry
weight (DW)) of tomato plants cv. F-144 exposed to transient water stress. The bacterial suspension was applied once, one week after the plants were
transplanted. Watering was stopped 21 days after germination (indicated as 0 on the time scale) and resumed 33 after germination (indicated as 12 on
the time scale). Values are the means of 6 replicates S.E.

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S. Mayak et al. / Plant Science 166 (2004) 525530

ARV8 significantly promoted the growth of pepper plants

as manifested in the higher fresh and dry weights attained
by the bacterially treated pepper plants. The ratio of bacterial treated/bacterial non-treated was higher in tomato plants
compared with pepper plants. It should be realized however
that the comparison is of limited value only (experimental
protocol varied slightly). At present its value is suggestive
and it needs to be improved.
In a more detailed study of the response of seedlings to a
reduction in water availability, the fresh weight was monitored at 0, 6, 12, 17 and 23 days following the onset of the
stress; at 17 and 23 days, the watering had been resumed.
Although the seedlings were not supplied with water for 6
days their fresh weight increased (Fig. 1). This probably reflects the fact that during this period the plants were able
to secure water from the growth medium. The observed increase in RWC supports this suggestion (Fig. 2). The increase in fresh weight was similar in control and stressed
seedlings. On the other hand, no change in fresh weight in
the stressed seedlings was observed during the next 6 days,
during which water was not supplied.
After 12 days of drought stress watering was resumed but
the measurements demonstrate that after five additional days
(day 17 after the initiation of the stress) the fresh weight did
not increase in the previously stressed seedlings. After 6 additional days (that is 23 days after the initiation of the stress)
a small increase in the fresh and dry weight was measured in
the previously stressed seedlings. In contrast, a continuous
and significant increase in the fresh and dry weights of the
stressed seedlings that were treated with the bacterial strain
A. piechaudii ARV8 was observed. This increase in weight
also occurred during the resumption of the water supply. At
the end of the experiment (indicated as day 23 in Figs. 1

and 2) the fresh weight of the bacterially treated seedlings

was about twice that of control plants. Not surprisingly,
the changes in the dry weights of seedlings paralleled the
changes in seedling fresh weight (Fig. 1B). The fresh weight
attained by the bacterially treated, but not stressed seedlings
was the highest compared with the other treatments.
During water deprivation the RWC in the plants declined
in stressed plants, both treated and not treated with bacteria. The lowest value was measured at day 12 of the stress
(Fig. 2). This reflects an increasing water stress [23]. When
watering was resumed, the RWC increased, attaining higher
values in the bacterially treated plants. In the presence of
the bacterium, and independent of whether or not the plants
were stressed, by 23 days after the onset of the stress the
RWC was significantly greater in the presence than in the
absence of the bacterium. Moreover, while the bacterium did
not help the seedlings to maintain their RWC during periods
of drought, the presence of the bacterium greatly facilitated
the recovery of the seedlings upon re-watering.
3.1. Effect on ethylene production
Nine days after seedlings were exposed to water stress
ethylene production began to increase. Thereafter, the rise
in the rate of ethylene production continued to a value of
28 nl h1 at 13 days (Fig. 3). By comparison, control (hydrated) seedlings reached a rate of production of 1.5 nl h1 .
By inoculating seedlings with A. piechaudii ARV8, and then
exposing them to water stress, the rise in the rate of ethylene
production occurred later and the rise in the rate of ethylene production was smaller than in stressed seedlings. At
13 days the rate of ethylene production has reached a lower
value of 6.1 nl h1 (Fig. 3).

Fig. 2. The effect of the ACC deaminase-containing plant promoting bacterium A. piechaudii ARV8 on the relative water content (RWC) of tomato
plants cv. F-144 exposed to transient water stress. The bacterial suspension was applied once, 1 week after the plants were transplanted. Watering was
stopped 21 days after germination (indicated as 0 on the time scale) and resumed 33 after germination (indicated as 12 on the time scale). Values are
the means of 6 replicates S.E.

S. Mayak et al. / Plant Science 166 (2004) 525530

Fig. 3. The effect of ACC deaminase-containing plant growth-promoting

bacteria on the rate of ethylene production by tomato seedlings. The
time axis indicates days after germination. Water stress was initiated by
removal of water eight days after germination. Values are means of 6
replicates S.E. (when larger than the symbol).

529

The bacterium is envisioned as affecting the production

of ethylene, which in turn influences the factors described
above. This suggestion is supported by earlier observations
that ethylene reduces the fluidity of membranes [24,25], influences phospholipid turnover in membranes [26], increases
leakage of solutes from plant cells [27,28] and suppresses
elongation of roots [22,29,30]. In addition stress is known to
stimulate ethylene production [25,31,32]. Thus, under stress
conditions, ACC deaminase-containing bacteria that restrain
the production of ethylene (Fig. 3) may be effective in alleviating a portion of the stress effect. However, other elusive
factors evolved in bacteria populating ecological sites, where
water is scarce and dry periods occur frequently, may also
interact with plants to increase resistance to water stress.
The precise mechanisms notwithstanding, the use of
plant growth promoting bacteria that decrease the damage
to plants that occurs under drought conditions is a potentially important adjuvant to agricultural practice in locales
where drought is endemic.

4. Discussion
It has been suggested that bacteria containing ACC deaminase activity should all act to reduce the level of stress
ethylene and thus confer resistance to various stresses [3].
Indeed this suggestion is supported by the results of the
present experiments (Tables 1 and 2) and also those that
were reported previously demonstrating increased resistance to salt stress [1], flooding stress [15], heavy metal
stress [14] and pathogen stress [16].
We have hypothesized that those bacteria populating sites
where water is limited and repeated dry periods occur frequently, are likely to be able to better promote plant growth
compared with plant growth promoting bacteria isolated
from sites where water sources are abundant. The results of
our study support this hypothesis. The seedlings treated with
A. piechaudii ARV8, a bacterial strain isolated from an arid
site, were significantly larger than the seedlings treated with
a bacterium, P. putida GR12-2, that was originally isolated
from the rhizosphere of grasses in the High Canadian Arctic
[20] where water is abundant.
In the present experiments, the bacterially treated plants
that were exposed to water stress continued to accumulate
plant biomass (Fig. 1), although water availability was reduced (reflected in lowered RWC values), especially during
the 6 days between 6 and 12 days of the stress period (Fig. 2).
Apparently, the underlying processes causing a gain in plant
biomass were stimulated in bacterially treated plants, resulting in a continued increase in dry weight to a greater extent than in control hydrated plants. A similar change was
observed in fresh weight except for the second part of the
stress period (between 6 and 12 days) (Fig. 1), when gain in
fresh weight was stopped. In the absence of bacterial treatment, the growth processes were disturbed during the period of reduced water availability resulting in cessation of
growth, and this inability to grow continued when watering
was resumed (Fig. 1).

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