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FEMS Microbiology Ecology 35 (2001) 137^144
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Biological activity and colonization pattern of the
bioluminescence-labeled plant growth-promoting bacterium
Kluyvera ascorbata SUD165/26
Wenbo Ma, Kelly Zalec, Bernard R. Glick *
Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1
Received 9 October 2000; received in revised form 1 December 2000; accepted 3 December 2000
Abstract
Kluyvera ascorbata SUD165/26 is a spontaneous siderophore-overproducing mutant of K. ascorbata SUD165, which was previously
isolated from nickel-contaminated soil and shown to significantly enhance plant growth in soil contaminated with high levels of heavy
metals. To develop a better understanding of the functioning of K. ascorbata SUD165/26 in the environment, and to trace its distribution in
the rhizosphere, isolates of this bacterium were labeled with either green fluorescent protein or luciferase. When the plant growth-promoting
activities of the labeled strains were assayed and compared with the activities of the unlabeled strain, none of the monitored parameters had
changed to any significant extent. When the spatial colonization patterns of the labeled bacteria on canola roots were determined after seed
application, it was observed that the bacterium was tightly attached to the surface of both roots and seeds, and formed aggregates. The
majority of the bacterial population inhabited the upper two thirds of the roots, with no bacteria detected around the root tips. ß 2001
Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Bioluminescence labeling; Heavy metal; Phytoremediation; Plant growth-promoting rhizobacterium; Kluyvera ascorbata SUD165/26
1. Introduction
Pollution by heavy metals as a consequence of burning
of fossil fuels, mining, smelting, municipal wastes, fertilizers, pesticides and sewage has caused severe environmental problems [1]. Phytoremediation, or the use of plants to
remove, destroy or sequester hazardous substances from
the environment [2,3], has received considerable attention
recently because, unlike traditional remediation of these
environmental contaminants which usually involves expensive excavation and removal of soil for treatment, it is
perceived to be environmentally friendly and relatively
low cost. For example, metal-tolerant and metal-accumulating plants have been used to treat metal mine tailings
and waste piles [2]. Unfortunately, however, high levels of
heavy metals are still toxic to these metal-tolerant plants,
and lead to low levels of plant biomass, and therefore
ine¤cient phytoremediation.
Plant growth-promoting rhizobacteria (PGPR) are free-
* Corresponding author. Tel. : +1 (519) 888-4567 ext. 2058 ;
Fax: +1 (519) 746-0614; E-mail: [email protected]
living soil bacteria found on or near the roots of plants
and can exert bene¢cial e¡ects on plant growth either directly or indirectly [4]. Directly, PGPR may provide plants
with compounds synthesized by bacteria, such as ¢xed
nitrogen or phytohormones; they may facilitate the uptake
of nutrients such as phosphorus and iron, or they may
synthesize the enzyme 1-aminocyclopropane-1-carboxylate
(ACC) deaminase which lowers plant ethylene levels [5,6].
Indirectly, PGPR facilitate plant growth by preventing or
decreasing the deleterious e¡ects of the pathogenic microorganisms in the rhizosphere [7].
Kluyvera ascorbata SUD165 was previously isolated
from a nickel-contaminated soil sample and shown to signi¢cantly enhance plant growth in the presence of heavy
metals, such as zinc, lead and nickel [1,8]. K. ascorbata
SUD165/26 is a spontaneous siderophore-overproducing
mutant of K. ascorbata SUD165 that is more e¤cient
than K. ascorbata SUD165 in promoting plant growth in
the presence of high levels of heavy metals [8]. It has been
proposed that the ACC deaminase activity and a high
level of siderophores are the main reasons that this bacterium stimulates plant growth in the presence of heavy
metals [8]. The bacterial ACC deaminase lowers `stress'
0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 0 0 ) 0 0 1 2 1 - 5
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W. Ma et al. / FEMS Microbiology Ecology 35 (2001) 137^144
ethylene [9] production induced by heavy metals, thereby
preventing the inhibition of plant growth by stress ethylene. Heavy metals can induce iron de¢ciency in plants,
and the application of iron salts to the plants reduces
the severity of nickel toxicity [10]. Bacterial siderophores
can form a tight complex with iron in the environment,
and the iron^siderophore complex may be taken up by the
plants in the immediate vicinity, thereby providing the
plants with iron regardless of the presence of nickel or
any other heavy metal [11].
Several other traits of PGPR, such as soil persistence
and root colonization, are essential for its functioning.
Moreover, it is necessary to understand the functioning
of a plant growth-promoting bacterium in the environment in order to develop better remediation strategies
[12]. Thus, K. ascorbata SUD165/26 was labeled with
green £uorescent protein (GFP) from the jelly¢sh Aquoria
victoria, and with luciferase (Lux) from the bacterium Vibrio harveyi. GFP and Lux are both very sensitive, easily
detected and can be quanti¢ed with a spectro£uorimeter
or a luminometer [13,14], thereby providing a simple and
accurate means of monitoring and localizing selected bacteria in a complex environment.
In the work reported here, GFP and Lux genes were
introduced into K. ascorbata 165/26, and maintained on
a plasmid or a transposon, respectively. ACC deaminase
activity and siderophore levels, as well as the bacterial
growth rate and persistence in nickel-contaminated soil
of the labeled bacteria, were monitored. The plant
growth-promoting abilities with and without nickel were
also tested in both growth pouch and pot assays. In addition, spatial colonization patterns of the bacteria along
canola roots were determined.
2. Materials and methods
2.1. Media, plasmids and bacterial strains
K. ascorbata SUD165/26 was grown in nutrient broth
(Difco Laboratories, Detroit, MI, USA) or in M9 minimal
medium [19] at 30³C. Escherichia coli strains were grown
in Lennox L broth (Gibco BRL Life Technologies, Paisley, UK) or in M9 minimal medium supplemented with
casamino acids at 37³C.
2.2. Construction of GFP and Lux-labeled K. ascorbata
SUD165/26
K. ascorbata SUD165/26/p519gfp was constructed by
triparental mating of E. coli DH5K/p519gfp, E. coli
HB101/pRK2013 and K. ascorbata SUD165/26 [17]. Nutrient agar supplemented with kanamycin (50 Wg ml31 )
and ampicillin (100 Wg ml31 ) was used to select the transconjugants.
Plasmid pJQ855 contains luxAB genes under the transcriptional control of the Enterobacter cloacae UW4 ACC
deaminase gene promoter region ; this promoter needs
ACC to be induced. The ACC deaminase promoter
(pacd) and the luxAB genes were excised from pJQ855
by SmaI and PvuII digestion and inserted into the unique
SphI site of the mini-Tn5 Km1 vector (Fig. 1). Mini-Tn5
Km1: :pacd-lux was introduced into K. ascorbata SUD165/
26 by triparental mating [17], and the cassette which includes pacd-luxAB and kanamycin resistance genes was
subsequently integrated into the host chromosomal
DNA. The presence of pacd-luxAB in the chromosomal
DNA was ascertained by Southern hybridization (data
not shown).
The induction of the luxAB genes by ACC in K. ascorbata SUD165/26: :pacd-lux was monitored by using a luminometer. The amount of luminescence was proportional
to the ACC concentration, and was maximal when 2 mM
ACC was added. Higher concentrations of ACC did not
result in any additional increase in luminescence. Therefore, 2 mM ACC was used to induce the luxAB genes in
subsequent experiments.
The bacterial strains and plasmids used in this study are
shown in Table 1.
Table 1
Plasmids and bacteria strains used in the study
Bacterial strain or plasmid
Bacterial strains:
K. ascorbata SUD165/26
K. ascorbata SUD165/26/p519gfp
K. ascorbata SUD165/26 : :pacd-lux
E. coli DH5K/p519gfp
E. coli CC118
E. coli S17.1/mini-Tn5 Km1
E. coli HB101/pRK2013
Plasmids :
pJQ855
mini-Tn5 Km1 : :pacd-lux
Relevant characteristics
Source or reference
heavy metal tolerance, Apr
Apr , GFP‡ , Kmr
Apr ,Lux‡ , Kmr
GFP‡ , gfp mut2 cloned downstream of the lac promoter, Kmr
Vpir lysogen
Kmr , tnp*, ori (R6K)
Kmr , mob‡
[1]
this study
this study
ATCC87452, [13]
[15]
[16]
[17]
pQF70 derivative, Lux‡ , luxAB genes cloned downstream of the
E. cloacae UW4 ACC deaminase gene promoter
Kmr , tnp*, Lux‡
[18,14]
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W. Ma et al. / FEMS Microbiology Ecology 35 (2001) 137^144
139
0.2 mM and the soil was autoclaved; 1 ml of bacterial
suspension was added and thoroughly mixed with the soil
in each of the cups. The cups were placed in a growth
chamber maintained at 25³C; soil samples from each treatment were removed weekly for 6 weeks. For the enumeration of bacteria in the soil, 1 g of soil sample was taken from
each cup and suspended in 10 ml of sterile 0.85% NaCl.
After being gently vortexed, the soil suspensions were diluted and plated onto nutrient agar (50 Wg ml31 kanamycin
was added for the labeled strains). The plates were incubated at 30³C, and colonies were counted after 18 h.
2.5. Determination of ACC deaminase activities and
siderophore levels
The ACC deaminase activity of cell extracts was determined by the method of Honma and Shimomura [20]
which measures the amount of K-ketobutyrate that resulted from the deamination of ACC. Siderophores secreted to the growth medium were detected and quanti¢ed
by the method described by Schwyn and Neilands [21].
Fig. 1. Schematic representation of the construction of the suicide plasmid used to introduce luxAB genes with the promoter of ACC deaminase gene from E. cloacae UW4 into K. ascorbata SUD165/26 to create
K. ascorbata SUD165/26: :pacd-lux. Key : pacd: promoter of ACC deaminase gene from E. cloacae UW4. luxAB: genes encoding luciferase
from V. harveyi. mob: mobility genes of plasmid RP4. tnp*: mutated
transposition gene of R6K. ori: origin of replication. 0 end and I end:
19-bp terminal repeats of Tn5. Ap: ampicillin resistance gene. Km: kanamycin resistance gene.
2.3. Preparation of bacterial suspension
K. ascorbata SUD165/26, K. ascorbata SUD165/26/
p519gfp and K. ascorbata SUD165/26: :pacd-lux were
grown overnight in nutrient broth at 30³C; 50 Wg ml31
kanamycin was added to the labeled strains. Cells were
collected, washed twice with sterile 0.85% NaCl and then
resuspended in sterile 0.85% NaCl. The cell density was
adjusted to an absorbance at 595 nm of 0.5 (equivalent to
approximately 7.4U108 cfu ml31 ) and used for seed inoculation and persistence assays.
2.4. Persistence assays
Each experiment included eight plastic cups (4.5
cmU4.5 cmU5.5 cm) each containing 10 g of pre-wetted
Pro-Mix `BX' general purpose growth medium (General
Horticulture, Inc., Red Hill, PA, USA). This medium includes sphagnum peat moss (75^85% by volume), perlite,
vermiculite, macronutrients (calcium, magnesium, nitrogen, phosphorus, potassium, sulfur), micronutrients (boron, copper, iron, manganese, molybdenum, zinc), dolomitic limestone, calcite limestone, and a wetting agent.
The pH was approximately 6.0. Nickel was added to the
soil in the form of NiCl2 to a ¢nal concentration of
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2.6. Seed inoculation
For growth pouch and pot assays, canola seeds (Brassica rapa cv. Reward), kindly provided by Dr. Gerry
Brown (Ecosoil Systems, Inc., San Diego, CA, USA),
were surface-sterilized by soaking in 1.5% (v/v) sodium
hypochlorite for 10 min, and then thoroughly rinsed
with sterile distilled water. These seeds were then incubated with either 0.85% NaCl as a control, or with a bacterial suspension (absorbance at 595 nm of 0.5) for 1 h at
30³C [1].
2.7. Growth pouch assays
Seed-pack growth pouches (125U157 mm, Mega International, MN, USA) containing 10 ml distilled water or
3 mM NiCl2 solution were autoclaved. Six to eight inoculated canola seeds were placed in each of the pouches,
and then the pouches were incubated upright in a plastic
tray. The tray was covered by Saran Wrap1 and partially
¢lled with water, just su¤cient to cover the bottom in
order to prevent loss of moisture. The pouches were incubated in a growth chamber at 25³C with a 12-h photoperiod and a light intensity of 100 Wmol m32 s31 for 5 days
before the lengths of roots were measured [1].
2.8. Growth assays
Canola seeds inoculated with bacteria were sown in
plastic pots (bottom diameter = 145 mm, top diameter = 205 mm, height = 150 mm) ¢lled with growth medium
Pro-Mix `BX'. The medium was prepared by mixing the
dry medium with 3 mM NiCl2 . Three inoculated canola
seeds were sown per pot and 10 pots were used in each
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W. Ma et al. / FEMS Microbiology Ecology 35 (2001) 137^144
treatment. The pots were kept in a greenhouse that ranged
from 20 to 25³C (night to day), and with a 16-h photoperiod using £uorescent supplementary lighting with a
light intensity of approximately 200 Wmol m32 s31 . The
plants were watered every other day for 25 days.
The plants were removed from the soil carefully and
washed extensively with water to remove soil particles.
Fresh weights of the plants were determined immediately,
and dry weights were made after they were oven-dried
overnight at 105³C.
Leaves were excised from the plants for chlorophyll and
protein assays. The chlorophyll content was measured by
the method of Hiscox and Israelstam [22], and the protein
assay was done as described by Burd et al. [8].
2.9. Statistical analysis
Data were analyzed by analysis of variance (ANOVA),
and treatment means were compared by Tukey's HSD
multiple comparison test to determine whether the treatments were signi¢cantly di¡erent between the control and
each other. All hypotheses were tested at the 95% con¢dence interval.
2.10. Determination of luminescence produced by
Lux-labeled bacteria
were ¢lled with unsterile Pro-Mix `BX' medium and rinsed
with tap water. Three inoculated seeds (as described in
growth pot assays) were sown in each pot and then grown
in a growth chamber at 25³C without watering. The seedlings were carefully removed from the soil after 3 days and
tapped lightly to remove adhering soil.
For the seeds inoculated with K. ascorbata SUD165/
26: :pacd-lux, roots were placed on M9 plates supplemented with 50 Wg ml31 kanamycin and 2 mM ACC
and then incubated at 30³C. After 18 h, n-decyl aldehyde
was swabbed inside the petri dish lids and the roots were
visualized by chemiluminescence and visible light imaging.
The roots of the seeds inoculated with K. ascorbata
SUD165/26/p519gfp were rinsed with sterile distilled water
to remove any bacteria that were not tightly attached to
the roots, and then placed on clean glass microscope
slides; a drop of mounting solution (80% glycerol,
1UPBS, 2.5% 1,4-diazabicyclo-[2.2.2]octane) was added
on top of each root and a cover slip was applied. The
bacteria attached to the roots were visualized by epi£uorescence microscopy and laser scanning confocal microscopy (Carl Zeiss, Jena, Germany) [23].
3. Results and discussion
K. ascorbata SUD165/26: :pacd-lux was grown overnight
at 30³C on M9 plates with 50 Wg ml31 kanamycin and
2 mM ACC, and the luminescent colonies were detected
by swabbing the inside of the petri dish lid with n-decyl
aldehyde (Sigma). A blue^green light was produced and
detected visually in a dark room [14] or by a chemoluminescence and visible light imaging system (Fluorchem1,
Alpha Innotech Co., San Leandro, CA, USA).
A luminometer (TD-20/20, Turner Designs, Sunnyvale,
CA, USA) was used to quantify the amount of luminescence produced by the bacteria. Cells from an overnight
culture grown in M9 minimal medium supplemented with
50 Wg ml31 kanamycin and 2 mM ACC were pelleted and
washed with 0.85% NaCl. Five Wl of n-decyl aldehyde was
added to 500 Wl of this cell suspension, mixed completely
and the light units produced were read from the luminometer immediately.
3.1. Growth rates of K. ascorbata SUD165/26 labeled with
GFP and Lux
The e¡ects of the presence of p519gfp and the mini-Tn5
Km1 transposon on the growth of K. ascorbata SUD165/
26 in both nutrient broth and M9 minimal medium were
determined. In both types of media, the three strains grew
at nearly identical rates. Although the original strain had a
lag phase that was about 1 h shorter than either of the
labeled strains, the doubling times of all three strains were
about 1.6 h in nutrient broth and 1.9 h in M9 minimal
medium.
2.11. Determination of green £uorescence produced by
GFP-labeled bacterium
Colonies of K. ascorbata SUD165/26/p519gfp were examined visually in a dark room using a long-wavelength
UV lamp, or by epi£uorescence microscopy [23].
2.12. Determination of spatial colonization patterns of
labeled bacteria on canola roots
Three pots of the size described in the growth assays
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Fig. 2. Persistence of K. ascorbata SUD165/26 and labeled strains in
sterile plant growth medium (Pro-Mix `BX' general purpose medium) in
the presence of 0.2 mM NiCl2 . The error bars represent the S.E.M. of
eight repeats.
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W. Ma et al. / FEMS Microbiology Ecology 35 (2001) 137^144
141
The longer lag phase and subsequent late initiation of
the exponential phase in the case of the labeled strains
may re£ect a metabolic load caused by the energy required
to synthesize and express the foreign genes, i.e. the kanamycin resistance gene, the GFP gene and the luciferase
genes [24]. When tested for genetic stability (data not
shown), both labeled strains completely maintained their
marker genes for more than 10 generations in the absence
of selective antibiotics. These results indicate that the
growth of K. ascorbata SUD165/26 was not signi¢cantly
a¡ected by the labeling.
3.2. Persistence of the bacteria in soil with nickel
K. ascorbata SUD165/26 and its derivatives were added
to soil that contained 0.2 mM nickel, and the numbers of
culturable bacteria in the soil were counted every week for
6 weeks (Fig. 2). The results showed that all three strains
survived well in soil in the presence of nickel. Although
di¡erences were observed in the number of bacteria that
could be recovered from the soil at various times, the
patterns of the numbers of the three strains over the
6-week period were similar: the number of bacteria decreased in the ¢rst 2 weeks, and then increased again.
From the third week, the number of bacteria decreased
gradually, no further increase was observed during the
next 4 weeks. Of the three strains, those that formed the
highest number of colonies were observed in the soil with
the unlabeled strain although the di¡erences were small.
Again, the metabolic burden from the introduced foreign
genes may contribute to the lower number of the labeled
strains.
3.3. ACC deaminase activities and siderophore levels of
K. ascorbata SUD165/26 strains
Previous results showed that K. ascorbata SUD165/26
has a relatively low level of ACC deaminase activity and
produces a high level of siderophores [1,8]. It is believed
that these characteristics contribute to the plant growthpromoting activity of this bacterium. The ACC deaminase
activities and siderophore levels were determined in the
labeled strains. The ACC deaminase activities of K. ascor-
Fig. 3. Growth-promoting e¡ect of K. ascorbata SUD165/26 and labeled
strains on root length of 5-day-old canola plants with and without
3 mM NiCl2 in gnotobiotic growth pouches. The error bars represent
the S.E.M. of 50 samples.
bata SUD165/26/p519gfp and K. ascorbata SUD165/
26: :pacd-lux were 20.9 þ 1.8 and 20.3 þ 2.3 nmol mg31
h31 , respectively, compared with 20.8 þ 0.8 nmol mg31
h31 for the unlabeled strain (n = 3, þ S.E.M.). The
amounts of siderophore secreted by the bacterial strains :
K. ascorbata SUD165/26, K. ascorbata SUD165/26/
p519gfp and K. ascorbata SUD165/26: :pacd-lux were
51.9 þ 2.7, 56.6 þ 0.2 and 55.7 þ 1.3 Wmol OD31 , respectively (n = 2, þ S.E.M.). In both instances the measured
values for the labeled strains were very similar to the values of the original strains.
3.4. The e¡ect of K. ascorbata SUD165/26 and labeled
strains on the growth of canola
Growth pouch assays were performed to assess the
plant growth-promoting activity of K. ascorbata
SUD165/26 and the labeled strains grown in the presence
or absence of nickel. The results showed that, either with
or without nickel, the bacteria signi¢cantly promoted root
elongation (Fig. 3). The root length decreased by more
than 30% in the presence of 3 mM nickel, and in the
presence of the bacteria, the length of canola roots reached
the level of untreated roots without nickel. This result was
consistent with the previous hypothesis that ACC deami-
Table 2
The e¡ects of K. ascorbata SUD165/26 and the labeled strains on the growth of 25-day-old canola in the soil with 3 mM of nickel
0.85% NaCl
0.85% NaCl +3 mM Ni
K. ascorbata SUD165/26+3 mM Ni
K. ascorbata SUD165/26 : :pacd-lux +3 mM Ni
K. ascorbata SUD165/26/p519gfp +3 mM Ni
Fresh weight
(g/plant)
Dry weight
(g/plant)
Chlorophyll
(mg g31 leaf)
Protein
(mg g31 leaf)
2.55 þ 0.20a
0.83 þ 0.07b
1.36 þ 0.16c
1.25 þ 0.10c
1.27 þ 0.15c
0.162 þ 0.011a
0.066 þ 0.008b
0.098 þ 0.009c
0.096 þ 0.007c
0.095 þ 0.010c
2.27 þ 0.29a
1.39 þ 0.19b
2.21 þ 0.31a
1.76 þ 0.24ab
2.10 þ 0.34a
2.83 þ 0.34a
2.40 þ 0.62a
2.58 þ 0.55a
2.46 þ 0.50a
2.71 þ 0.82a
Superscripted letters indicate values that are either statistically signi¢cantly di¡erent (when the letters are di¡erent) or not (when the letters are the
same) with the 0.85% NaCl treatment. The sample size for the fresh weight and dry weight were 30 ( þ S.E.M.), and for the chlorophyll content and
protein content were 15 ( þ S.E.M.).
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nase activity in the bacteria can promote root elongation
by lowering the ethylene level in seedlings. In addition,
since there was su¤cient iron in the seeds for the development of these 5-day-old seedlings, the bacterial siderophores were unlikely to have any e¡ect on the development of canola seedlings in the pouch assay [1].
The e¡ect of K. ascorbata SUD165/26 and labeled
strains on the growth of canola for 25 days in soil with
3 mM nickel was also monitored (Table 2). It was obvious
that nickel in the soil had an inhibitory e¡ect on the
growth of canola plants. The dramatic reduction in the
fresh and dry weight was evidence of decreased plant
growth. A decrease in leaf chlorophyll was also observed,
probably because nickel exerts an e¡ect on iron metabolism and localization in plants [10]. When the bacteria
were added, the results indicated that all of the bacterial
strains enhanced plant growth in soil that contained nickel
and partially relieved the phytotoxicity: the fresh and dry
weights increased to some degree and the chlorophyll content of leaves attained a level as high as that in leaves not
treated with nickel. In addition, the protein content was
not signi¢cantly a¡ected by either nickel or added bacteria. In all cases, the labeled bacteria performed nearly as
well as the original strain except that strain K. ascorbata
SUD165/26: :pacd-lux was unable to completely restore
the chlorophyll content to the level observed with the unlabeled strain.
3.5. Spatial colonization patterns of K. ascorbata
SUD165/26
Fig. 4. Spatial distribution of K. ascorbata SUD165/26: :pacd-lux on the
3-day-old canola root by chemoluminescence and visible light imaging
system. (A) is the root which had been placed on M9 minimal medium
supplemented with 50 Wg ml31 kanamycin and 2 mM ACC overnight at
30³C to allow the bacteria which were attached to the root to grow and
the luxAB genes integrated in the chromosome of the bacteria to be induced. The bacteria attached to the root could be seen growing on the
medium. (B) is the luminescence of the root in (A) detected in the dark
after exposure for 6 min with a chemoluminescence digital imager. The
bacteria K. ascorbata SUD165/26: :pacd-lux attached along the root produced white light catalyzed by luciferase when substrate n-decyl aldehyde was added. The light dot at the top was the luminescence produced by the bacteria attached to the seed coat.
FEMSEC 1210 29-3-01
Binding to plant roots is usually necessary before a
plant growth-promoting bacterium can have a bene¢cial
e¡ect on plant growth. Therefore, it is important to know
whether a bacterium is able to attach to plant roots; the
localization of that attachment might help to understand
the functioning of the bacterium. Bioluminescence labeling
makes it possible to monitor a selected bacterium in a
complex environment easily and precisely [25].
In this study, the green £uorescence and luminescence
were detected on 3-day-old canola roots inoculated with
K. ascorbata SUD165/26/p519gfp and K. ascorbata
SUD165/26: :pacd-lux by seed application. It was clear,
from chemoluminescence imaging (Fig. 4), that the bacteria can tightly attach to both the seed coat and the upper
two thirds of the root. In fact, the majority of the bacterial
population was localized on the upper two thirds of the
root, and there was no detectable luminescence found in
the region close to the root tip.
Previous studies showed that bacteria tend to form aggregates or microcolonies on plant surfaces such as roots,
leaves or seeds [26,27] and even on inner root tissues [28].
It was observed in this study that K. ascorbata SUD165/
26/p519gfp formed aggregates on the surface of canola
roots, but no bacteria were observed in the inner root
tissue (Fig. 5). In addition, it was observed that more
bacteria colonized the upper part of the root, and no bacteria were found around the root tip.
The plant growth-promoting bacterium K. ascorbata
SUD165/26 was genetically labeled with GFP or Lux.
Both of the labeled strains showed similar growth rates
and levels of persistence when compared to the original
strain. In addition, most, if not all, of the abilities of the
original strain to promote canola growth in the presence
and absence of nickel were retained. Furthermore, the labeled strains were used to determine the localization of the
bacterium along canola roots: the labeled strains were visualized, microscopically and macroscopically, by £uores-
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W. Ma et al. / FEMS Microbiology Ecology 35 (2001) 137^144
143
cence and luminescence. These results suggest that the labeled strains hold some promise for understanding the
functioning of plant growth-promoting bacteria under a
variety of environmental conditions including the presence
of heavy metals; as well, the labeled strains can provide
useful information regarding the phytoremediation of
heavy metal-contaminated soil.
Acknowledgements
We are grateful to CRESTech; Inco, Ltd. ; and NSERC
Canada for providing funds in support of this research.
References
Fig. 5. Green £uorescence produced by K. ascorbata SUD165/26/
p519gfp was detected by laser scanning confocal microscopy. The microscopy used in this study was a Zeiss Axiovert 135 inverted microscope, equipped with 16U and 40U Neo£uor objectives and the LSM
410 confocal attachment. The excitation wavelength was 488 nm, and
the emission wavelength was 568 nm. The 3-day-old canola roots which
were seed inoculated with K. ascorbata SUD165/26/p519gfp were excised
from the shoots and placed on clean slides, one drop of mounting medium was added above the roots before examination. (A) is the image
from the upper part of the canola roots. The green £uorescence was
produced from the GFP produced by the bacteria and the root tissue
was red in color. (B) is the image from the lower part of the canola
roots. The bacteria were seen attached to the surface of roots including
the root hair, but the number and the density of the bacteria were
much lower than that from the upper part of the roots (A).
FEMSEC 1210 29-3-01
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