PHYSIOLOGIA PLANTARUM 118: 1015.

2003
Printed in Denmark all rights reserved

Copyright # Physiologia Plantarum 2003

ISSN 0031-9317

Technical focus

Methods for isolating and characterizing ACC deaminase-containing

plant growth-promoting rhizobacteria
Donna M. Penrose and Bernard R. Glick*
Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
*Corresponding author, e-mail: glick@sciborg.uwaterloo.ca
Received 15 August 2002; revised 5 November 2002

One of the major mechanisms utilized by plant growthpromoting rhizobacteria (PGPR) to facilitate plant growth and
development is the lowering of ethylene levels by deamination
of 1-aminocyclopropane-1-carboxylic acid (ACC) the immediate precursor of ethylene in plants. The enzyme catalysing this
reaction, ACC deaminase, hydrolyses ACC to a-ketobutyrate
and ammonia. Several bacterial strains that can utilize ACC
as a sole source of nitrogen have been isolated from rhizosphere soil samples. All of these strains are considered to be
PGPR based on the ability to promote canola seedling root

Mechanisms used by plant growth-promoting

rhizobacteria
The bacteria that provide some benefit to plants are of
two general types: those that form a symbiotic relationship, which involves formation of specialized structures
or nodules on host plant roots, and those that are freeliving in the soil; the latter are often found near, on or
even within plant tissues (Kloepper et al. 1988, van Peer
and Schipper 1989, Frommel et al. 1991). Beneficial freeliving soil bacteria are generally referred to as plant
growth-promoting rhizobacteria (PGPR) and are found
in association with the roots of many different plants.
Although numerous free-living soil bacteria are considered to be PGPR, not all bacterial strains of a particular
genus and species have identical metabolic capabilities
and interactions with plants. For example, some Pseudomonas putida strains actively promote plant growth
whereas others have no measurable effect on plants.

elongation under gnotobiotic conditions. The treatment of

plant seeds or roots with these bacteria reduces the amount
of ACC in plants, thereby lowering the concentration of ethylene. Here, a rapid procedure for the isolation of ACC
deaminase-containing bacteria, a root elongation assay for
evaluating the effects of selected bacteria on root growth,
and a method of assessing bacterial ACC deaminase activity are described in detail. This should allow researchers
to readily isolate new PGPR strains adapted to specific
environments.

Conceptually, PGPR can have an impact on plant

growth and development in two different ways: indirectly
or directly. The indirect promotion of plant growth occurs
when bacteria decrease or prevent some of the deleterious
effects of a phytopathogenic organism by one or more
mechanisms. On the other hand, the direct promotion of
plant growth by PGPR generally entails providing the
plant with a compound that is synthesized by the bacterium or facilitating the uptake of nutrients from the environment (Glick 1995, Glick et al. 1999).
Since many of the chemical agents that are used to
prevent disease symptoms in plants are both hazardous
to animals and humans, and can persist and accumulate in
natural ecosystems, chemicals are being replaced with
environmentally benign biological approaches to indirectly
promote plant growth including the use of biocontrol
PGPR. At present, there are fewer than 20 different biocontrol PGPR strains that are commercially available.

Abbreviations ACC, 1-aminocyclopropane-1-carboxylate; Pseudomonas Agar F (PAF); PGPR, plant growth-promoting rhizobacteria.

10

Physiol. Plant. 118, 2003

However, this number should increase as new biocontrol

strains are isolated using any one of a variety of available
screening procedures (Berg 1996, Eden et al. 1996, Gould
et al. 1996, Putcha and Allen 1997), and as superior,
genetically engineered, biocontrol strains are developed.
Traits associated with the biocontrol of plant pathogens
include: (1) antibiotic synthesis; (2) secretion of ironbinding siderophores to obtain soluble iron from the
soil and provide it to a plant, and thereby deprive fungal
pathogens in the vicinity of soluble iron (Neilands and
Leong 1986, Dowling et al. 1996); (3) production of low
molecular weight metabolites, such as hydrogen cyanide,
with antifungal activity (Dowling and OGara 1994); (4)
production of enzymes including chitinase, b-1,3glucanase, protease, or lipase, which can lyse some fungal
cells (Chet and Inbar 1994); (5) out-competing phytopathogens for nutrients and niches on the root surface
(Kloepper et al. 1988, OSullivan and OGara 1992, Loper
et al. 1997); and (6) lowering the production of (pathogen)
stress ethylene (Hyodo 1991) in plants with the enzyme
ACC deaminase (Glick et al. 1998, Penrose et al. 2001).
There are several ways in which different PGPR may
directly facilitate the proliferation of their plant hosts.
They may: (1) fix atmospheric nitrogen and supply it to
plants; (2) synthesize siderophores which can provide
iron to plants; (3) synthesize various phytohormones,
including auxins and cytokinins; (4) provide mechanisms
for the solubilization of minerals such as phosphorus;
and (5) synthesize enzymes that can modulate plant
growth and development (Brown 1974, Davison 1988,
Kloepper et al. 1988, Lambert and Joos 1989, Jacobson
et al. 1994, Patten and Glick 1996). A particular bacterium may promote plant growth and development using
any one, or more, of these mechanisms. Moreover, a
bacterium may utilize different traits at various times
during the life cycle of the plant. For example, following
seed germination a PGPR may lower the plants ethylene
concentration thereby decreasing the ethylene inhibition
of seedling root length (Glick et al. 1998). Once the
seedling has depleted the resources that are contained
within the seed, the same PGPR may help to provide
the plant with iron and phosphorus from the soil.
The impact of the mechanisms by which the bacterium
provides a compound or nutrient such as fixed nitrogen,
phosphorus or iron to the plant, varies considerably
depending upon the soil composition. Thus, PGPR
often have little or no measurable effect on plant growth
when the plants are cultivated in nutrient-rich soil and
grown under optimal conditions.
A number of PGPR contain the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (Jacobson
et al. 1994, Shah et al. 1997, Glick et al. 1998, Shah
et al. 1998) and this enzyme can cleave the plant ethylene
precursor ACC, and thereby lower the level of ethylene
in a developing or stressed plant. For many plants a
burst of ethylene is required to break seed dormancy
(Esashi 1991) but, following germination, a sustained
high level of ethylene would inhibit root elongation
(Jackson 1991). PGPR that contain the enzyme ACC
Physiol. Plant. 118, 2003

deaminase, when bound to the seed coat of a developing

seedling, act as a mechanism for ensuring that the
ethylene level does not become elevated to the point where
initial root growth is impaired. By facilitating the formation of longer roots, these bacteria may enhance the
survival of some seedlings, especially during the first
few days after the seeds are planted. In addition, plants
that are treated with ACC deaminase-containing PGPR
are dramatically more resistant to the deleterious effects
of stress ethylene that is synthesized as a consequence of
stressful conditions such as flooding (Grichko and Glick
2001), heavy metals (Grichko et al. 2000), the presence of
phytopathogens (Wang et al. 2000), and drought and
high salt (S. Mayak, T. Tirosh & B.R. Glick, submitted).
In each of these cases the ACC deaminase-containing
PGPR markedly lowered the level of ACC in the stressed
plants thereby limiting the amount of stress ethylene
synthesis and hence the damage to the plant. These
bacteria are beneficial to plant growth since in the natural environment plants are often subjected to ethyleneproducing stresses. However, it should be emphasized
that ACC deaminase-containing PGPR facilitate plant
growth to a much greater extent with plants that are
ethylene sensitive such as canola, peppers and tomatoes.
It is expected that this activity will be useful in both
agricultural and horticultural settings, as well as in environmental clean-up (i.e. phytoremediation) protocols.

Selection of bacterial strains that contain ACC

deaminase
Prior to the discovery of the role of ACC deaminase in
the functioning of PGPR, researchers who wanted to
isolate new PGPR strains utilized a long and tedious
approach (Lifshitz et al. 1987, Mew et al. 1994,
Pozdnyakov 1994, Press and Kloepper 1994, Slininger et al.
1994). In brief, this entailed isolating a large number of
bacterial strains (often hundreds or even thousands)
from the soil around the roots of particular (usually)
crop plants. These bacteria were then tested using any
one of a number of biological assays (including the
gnotobiotic growth pouch assay described later). The
simplest of these screens entailed testing the isolated bacteria
for biocontrol activity against one or more fungal pathogens. The easiest way to do this is to test each bacterial
strain for antibiosis activity on agar plates. Other screening procedures test the ability of isolated bacterial strains
to protect plants against damage by certain fungal or
bacterial pathogens. While these procedures are effective,
they are both time consuming and limited in that they only
select for certain types of biocontrol strains and are
completely unable to select for bacteria that promote
plant growth directly. Typically, bacteria that promote
plant growth directly are selected from a large number of
soil bacteria by testing each bacterial strain for its ability
to promote plant growth, either in growth pouches or in
soil, a process that requires growing a large number of
plants for each strain that is being assessed.
11

As an alternative to the existing procedures for isolating new PGPR, which often took several months or more
before a suitable strain could be identified, a rapid and
novel procedure for the isolation of ACC deaminasecontaining bacteria (which, by definition, are able to
promote plant growth) was developed. Initially, this
technique was used to identify and isolate seven new
PGPR strains (Glick et al. 1995). These bacterial strains
were isolated from seven soil samples collected during
late summer in Waterloo, Ontario, Canada and various
locations in California, USA from the rhizosphere of
seven different plants. All seven strains were isolated
within the same 2-week period. Originally, these strains
were designated as Pseudomonas sp., but were subsequently re-classified following fatty acid analysis (Shah
et al. 1997). Four of the strains were Enterobacter
cloacae, two were Pseudomonas putida and one was
Pseudomonas fluorescens.
Our method of isolating PGPR entails screening soil
bacteria for the ability to use ACC as a sole nitrogen
source, a trait that is a consequence of the presence of the
activity of the enzyme, ACC deaminase. One gram of
soil soil in close proximity to plant roots are generally
enriched in ACC deaminase-containing bacteria is
added to 50 ml sterile medium containing (per litre) 10 g
proteose peptone, 10 g casein hydrolysate, 1.5 g anhydrous MgSO4, 1.5 g K2HPO4 and 10 ml glycerol (PAF
medium) in a 250-ml flask. The flask and its contents are
incubated in a shaking water bath (200 r.p.m.) at a temperature between 25 and 30 C. After 24 h, a 1-ml aliquot
is removed from the growing culture, transferred to 50 ml
of sterile PAF medium in a 250-ml flask and incubated at
200 r.p.m. in a shaking water bath for 24 h, at either 25
or 30 C, the same temperature as the first incubation.
Following these two incubations, the population of pseudomonads and similar bacteria (such as Enterobacter spp.)
is enriched and the number of fungi in the culture is reduced.
A 1-ml aliquot is removed from the second culture and
transferred to a 250-ml flask containing 50 ml sterile
minimal medium, DF salts (Dworkin and Foster 1958)
(per litre): 4.0 g KH2PO4, 6.0 g Na2HPO4, 0.2 g
MgSO4
7H2O, 2.0 g glucose, 2.0 g gluconic acid and
2.0 g citric acid with trace elements: 1 mg FeSO4
7H2O,
10 mg H3BO3, 11.19 mg MnSO4
H2O, 124.6 mg
ZnSO4
7H2O, 78.22 mg CuSO4
5H2O, 10 mg MoO3,
pH 7.2 and 2.0 g (NH4)2SO4 as a nitrogen source. In
our laboratory, the DF minimal medium is prepared as
follows: (1) the trace elements (10 mg H3BO3, 11.19 mg
MnSO4
H2O, 124.6 mg ZnSO4
7H2O, 78.22 mg
CuSO4
5H2O, and 10 mg MoO3) are dissolved in 100 ml
sterile distilled water and then stored in the refrigerator
for up to several months; (2) FeSO4
7H2O (100 mg) is
dissolved in 10 ml sterile distilled water and is stored in
the refrigerator for up to several months; (3) all of the
other ingredients including 4.0 g KH2PO4, 6.0 g
Na2HPO4, 0.2 g MgSO4
7H2O, 2.0 g glucose, 2.0 g gluconic acid, 2.0 g citric acid, 2.0 g (NH4)2SO4 and 0.1 ml of
each of the solutions of trace elements and FeSO4
7H2O
are dissolved in 1 l distilled water and autoclaved for no
12

more than 20 min. If this medium is prepared by dissolving one ingredient at a time, namely by not adding
another ingredient until the first one is completely dissolved, the final preparation should not contain a precipitate. Following an incubation of 24 h in a shaking
water bath at 200 r.p.m. at either 25 or 30 C, the same
temperature as the first incubation, a 1-ml aliquot is
removed from this culture and transferred to 50 ml sterile
DF salts minimal medium in a 250-ml flask containing
3.0 mM ACC (instead of (NH4)2SO4) as the source of
nitrogen. A 0.5 M solution of ACC (CalbiochemNovobiochem Corp., La Jolla, CA, USA), which is labile
in solution, is filter-sterilized through a 0.2-mm membrane
and the filtrate collected, aliquoted and frozen at 20 C.
Just prior to inoculation, the ACC solution is thawed
and a 300-ml aliquot is added to 50 ml sterile DF salts
minimal medium; following inoculation, the culture is
placed in a shaking water bath at 200 r.p.m. and grown
for 24 h at 2530 C.
Dilutions of this final culture are plated onto solid DF
salts minimal medium and incubated for 48 h at either 25
or 30 C, the same temperature as the previous incubations. These plates are prepared with 1.8% Bacto-Agar
(Difco Laboratories, Detroit, MI, USA), which has a
very low nitrogen content, and are spread with ACC
(30 mmol plate 1) just prior to use. Before streaking
with either a loopful of bacteria or an individual colony,
the ACC is allowed to dry fully. The inoculated plates
are incubated at the appropriate temperature no higher
than 35 C because all of the known ACC deaminases are
inhibited above this temperature for 3 days and the
growth on the plates is checked daily. Even when apparently nitrogen-free agar is used, and no additional source
of nitrogen is included in the medium, it is almost impossible to obtain plates with absolutely no bacterial growth
but it is possible to get plates with very, very light
growth.
In order to avoid isolating multiple copies of the same
bacterium, only a single colony from each soil sample is
selected for further testing. Each selected colony is tested
for the synthesis of siderophores (Schwyn and Neilands
1987), antibiotics (Wang et al. 2000) and indole acetic
acid (Glickmann and Dessaux 1995), as well as for plantgrowth stimulation and ACC deaminase activity. A variant of this procedure that utilizes plant extract medium
(Belimov et al. 2001) may be used to isolate ACC
deaminase-containing strains of Bacillus and other bacterial
strains not selected for by PAF medium.

Culture conditions for the induction of bacterial ACC

deaminase activity
The assessment of bacterial ACC deaminase activity and
root-growth enhancement both require bacterial growth
conditions that favour the induction of ACC deaminase.
The bacteria are cultured first in rich medium and then
transferred to minimal medium with ACC as the sole
source of nitrogen. Bacterial cells are grown to mid- up
Physiol. Plant. 118, 2003

to late-log phase in 15 ml rich medium, such as tryptic

soybean broth (TSB; Difco Laboratories) divided
between two culture tubes: each tube is inoculated with
5 ml of the appropriate strain. Cultures are incubated
overnight in a shaking water bath at 200 r.p.m. at the
temperature most suitable for the bacterial strain. The
accumulated biomass is harvested by centrifugation of
the contents of the combined tubes at 8000 g for 10 min
at 4 C. The supernatant is removed and the cells are
washed with 5 ml DF salts minimal medium. Following
an additional centrifugation for 10 min at 8000 g at 4 C,
the cells are suspended in 7.5 ml DF salts minimal medium in a fresh culture tube. Just prior to incubation, the
frozen 0.5 M ACC solution (prepared as described
above) is thawed, and an aliquot of 45 ml is added to
the cell suspension to obtain a final ACC concentration
of 3.0 mM. The bacterial cells are returned to the shaking
water bath to induce the activity of ACC deaminase at
200 r.p.m. for 24 h at the same temperature as the overnight incubation, either 25 or 30 C. The bacteria are
harvested by centrifugation at 8000 g for 10 min at 4 C.
The supernatant is removed, and the cells are washed by
suspending the cell pellet in 5 ml either 0.1 M Tris-HCl,
pH 7.6 if the cells are to be assayed for ACC deaminase
activity, or 0.03 M MgSO4 if they are to be used as a
bacterial treatment in the gnotobiotic root elongation
assay or the high-performance liquid chromatography
(HPLC) protocol for measuring ACC. Following centrifugation at 8000 g at 4 C for 10 min, the supernatant is
discarded. The washing procedure is repeated twice to
ensure that the pellet is free of the bacterial growth
medium. The pelleted cells are stored at either 20 C
for measurement of ACC deaminase activity or at 4 C
for seed treatment in the gnotobiotic root elongation
assay or HPLC measurement of ACC.

Gnotobiotic root elongation assay

The gnotobiotic root elongation assay is used as a
method of assessing the effect of various bacterial strains
on the growth of canola seedlings. Note that the roots of
other ethylene-sensitive plants also respond to ACC
deaminase-containing PGPR by producing longer roots
when assayed in this manner. Each of the several dozen
strains of ACC deaminase-containing soil bacteria that
have been isolated (Glick et al. 1995, Burd et al. 1998,
Belimov et al. 2001, S. Mayak, T. Tirosh & B.R. Glick,
submitted) was assayed by the root elongation assay and
shown to promote canola seedling growth under gnotobiotic conditions. The protocol described below is a
modification of the procedure developed by Lifshitz
et al. (1987) and is used to measure the elongation of
canola roots from seeds treated with different strains of
bacteria or chemical ethylene inhibitors. The bacterial
cell pellet, prepared as described above, is suspended in
0.5 ml sterile 0.03 M MgSO4 and then placed on ice. A
0.5-ml sample is removed from the cell suspension and
diluted eight to ten times in 0.03 M MgSO4; the absorbance of the sample is measured at 600 nm. This measPhysiol. Plant. 118, 2003

urement is used to adjust the absorbance, at 600 nm, of

the bacterial suspension to 0.15 with sterile 0.03 M MgSO4.
Seed-pack growth pouches (Northrup King Co.,
Minneapolis, MN, USA) are prepared for the gnotobiotic
assay of canola root elongation by adding 12 ml of distilled water to each growth pouch, wrapping them in
aluminium foil in groups of 10 that are placed in an
upright position to prevent water loss, and autoclaving
at 121 C for 15 min.
Canola seeds (Brassica campestris) are disinfected
immediately before use (Tomato seeds may also be used
in this assay.). The seeds (approximately 0.2 g per treatment) are soaked in 70% ethanol for 1 min in glass Petri
dishes followed by 1% sodium hypochlorite. After
10 min the bleach solution is suctioned off and the
seeds are thoroughly rinsed with sterile distilled water
at least five times. Each dish is incubated at room temperature for 1 h with the appropriate treatment: sterile
0.03 M MgSO4 (used as a negative control) or bacterial
suspensions in sterile 0.03 M MgSO4. Following the
incubation period, six seeds are placed in each growth
pouch with sterilized forceps and 10 pouches are used for
each treatment. The pouches are placed upright in a rack
(Northrup King Co.) such that the individual pouches
are not touching one another. Two empty pouches are
placed at the ends of each rack so that plants at the end
of a rack are not subjected to extremes of light or air
circulation. The racks are placed in a clean plastic bin
containing sterile distilled water to a depth of approximately 3 cm and covered loosely with clear plastic wrap
to prevent dehydration. The pouches are incubated in a
growth chamber (Conviron CMP 3244; Controlled
Environments Ltd, Winnipeg, MB, Canada) maintained
at 20 1 C with a cycle beginning with 12 h of dark
followed by 12 h of light (18 mmol m 2 s 1). The primary
root lengths are measured on the fifth day of growth and
the data are analysed. Seeds that fail to germinate 2 days
after they are sown (generally less than 5% of the seeds
utilized) are marked and the roots that subsequently
develop from these seeds are not measured. Typically,
the root lengths of treated canola seedlings are 4060%
(and sometimes 90120%) greater than the lengths of
untreated seedlings, with standard errors generally
around 5% of the length of the untreated seedling root.

Measurement of ACC deaminase activity

ACC deaminase activity is assayed according to a modification of the method of Honma and Shimomura
(1978) which measures the amount of a-ketobutyrate
produced when the enzyme ACC deaminase cleaves
ACC. The number of mmol of a-ketobutyrate produced
by this reaction is determined by comparing the absorbance at 540 nm of a sample to a standard curve of
a-ketobutyrate ranging between 0.1 and 1.0 mmol. A stock
solution of 100 mM a-ketobutyrate (Sigma-Aldrich Co., St
Louis, MO, USA) is prepared in 0.1 M Tris-HCl pH 8.5 and
stored at 4 C. Just prior to use, the stock solution is diluted
13

with the same buffer to make a 10-mM solution from which

a standard concentration curve is generated. Each in a series
of known a-ketobutyrate concentrations is prepared in a
volume of 200 ml, 300 ml of the 2,4-dinitrophenylhydrazine
reagent (0.2% 2,4-dinitrophenyl-hydrazine in 2 M HCl)
(Sigma-Aldrich Co.) is added, and the contents are vortexed
and incubated at 30 C for 30 min, during which time the
a-ketobutyrate is derivatized as a phenylhydrazone. The
colour of the phenylhydazone is developed by the addition
of 2.0 ml 2 M NaOH; after mixing, the absorbance of
the mixture is measured at 540 nm.
ACC deaminase activity is measured in bacterial
extracts prepared in the following manner. Bacterial cell
pellets, prepared as described above, are each suspended
in 1 ml of 0.1 M Tris-HCl, pH 7.6, and transferred to a
1.5-ml microcentrifuge tube. The contents of the 1.5-ml
microcentrifuge tube are centrifuged at 16 000 g for 5 min
and the supernatant is removed. The pellet is suspended
in 600 ml 0.1 M Tris-HCl, pH 8.5. Thirty microlitres of
toluene are added to the cell suspension and vortexed at
the highest setting for 30 s. At this point, a 100-ml aliquot
of the toluenized cells is set aside and stored at 4 C for
protein assay at a later time. The remaining toluenized
cell suspension is immediately assayed for ACC
deaminase activity.
All sample measurements are carried out in duplicate.
Two hundred microlitres of the toluenized cells are
placed in a fresh 1.5-ml microcentrifuge tube; 20 ml of
0.5 M ACC are added to the suspension, briefly vortexed, and then incubated at 30 C for 15 min. Following
the addition of 1 ml of 0.56 M HCl, the mixture is vortexed and centrifuged for 5 min at 16 000 g at room temperature. One millilitre of the supernatant is vortexed
together with 800 ml of 0.56 M HCl. Thereupon, 300 ml
of the 2,4-dinitrophenylhydrazine reagent (0.2% 2,4dinitrophenylhydrazine in 2 M HCl) are added to the
glass tube, the contents vortexed and then incubated at
30 C for 30 min. Following the addition and mixing of
2 ml of 2 N NaOH, the absorbance of the mixture is
measured at 540 nm.
The absorbance of the assay reagents including the
substrate, ACC, and the bacterial extract are taken into
account. After the indicated incubations, the absorbance
at 540 nm of the assay reagents in the presence of ACC is
used as a reference for the spectrophotometric readings; it
is subtracted from the absorbance of the bacterial extract
plus the assay reagents in the presence of ACC. The contribution of the extract, namely the absorbance at 540 nm
of extract and the assay reagents without ACC, is determined and subtracted from the absorbance value calculated above. This value is used to calculate the amount of
a-ketobutyrate generated by the activity of ACC deaminase. The range of 01.0 mmol of a-ketobutyrate absorbs in
the range of 01.6 at 540 nm. The lowest amount of a-ketobutyrate that can be precisely determined is 0.1 mmol.
Different organisms with a wide range of ACC deaminase activity can act as PGPR. A low level of ACC
deaminase activity, namely approximately  20 nmol
a-ketobutyrate mg 1 h 1 is sufficient to permit a bacterium
14

to grow on ACC and to act as a PGPR. Organisms with

higher levels of ACC deaminase activity, that is from 300
to 400 nmol a-ketobutyrate mg 1 h 1 do not necessarily
promote root elongation to any greater extent than the
strains that contain less enzyme activity.

Conclusions
Using the procedures described here it is relatively
straightforward to isolate and characterize new PGPR
strains. These bacteria may be used to facilitate the
growth of a variety of plants, especially under stressful
conditions such as flooding (Grichko and Glick 2001),
heavy metals (Burd et al. 1998), phytopathogens (Wang
et al. 2000), high salt (Mayak, Tirosh and Glick unpubl.
results) and drought (Mayak, Tirosh and Glick unpubl.
results). This activity is useful in both agricultural and
horticultural settings, as well as in environmental
cleanup (i.e. phytoremediation) protocols.
Finally, preliminary indications are that ACC
deaminase-containing PGPR strains are beneficial in the
field as well as under more controlled laboratory conditions.
Thus, some of the strains that we have isolated have
recently been used to promote the growth of rice seedlings and soybean plants in the field in China.
Acknowledgements The work described here was supported by
grants from the Natural Sciences and Engineering Research Council
of Canada to B.R.G.

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Edited by M. Griffith
Physiol. Plant. 118, 2003

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