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Early colonization of barley roots by Pseudomonas fluorescens studied by immunofluorescence technique and confocal laser scanning microscopy
FEMS Microbiology Ecology 23 (1997) 353^360
Early colonization of barley roots by Pseudomonas £uorescens studied by immuno£uorescence technique and confocal laser scanning microscopy Michael Hansen, Lene Kragelund, Ole Nybroe, Jan SÖrensen * Section of Genetics and Microbiology, Department of Ecology and Molecular Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C (Copenhagen), Denmark Received 29 October 1996; revised 24 April 1997; accepted 6 May 1997
Abstract
Highly specific polyclonal antibodies against two Pseudomonas fluorescens strains (DF57 and Ag1), which differed by approximately 10% of their utilizable substrates as tested in Biolog GN plates, were used for in situ labelling of bacterial cells colonizing barley roots grown in sterile soil. By using a confocal laser scanning microscope single bacteria of both strains could be detected on the roots. Seed-inoculated bacteria rapidly colonized the root surface (rhizoplane) by active migration; after 1 day the anterior part of the root was densely covered by bacteria occupying the crevices between epidermal root cells. As the roots became longer, this bacterial population in the rhizoplane formed long strings of closely associated cells. After 7 days, however, the rhizoplane population of string-forming cells was partially detached developing a patchy distribution along the root; a separate population of cells localized in the slime matrix (mucigel) surrounding the root was well developed at this time. Using two different fluorochromes attached to the antibodies, the two strains could be detected simultaneously in coinoculation experiments. The recordings, however, gave no indications of competition between the two strains during root colonization. Keywords : Root colonization; Pseudomonas £uorescens ; Immuno£uorescence; Confocal laser scanning microscopy
1. Introduction
In situ localizations of bacteria in the rhizosphere have been studied for several decades by using a number of di¡erent microscopic techniques. Light microscopy studies include direct observation of fresh material by phase contrast or interference light microscopy [1] or observations after staining of the * Corresponding author. Tel.: +45 35 28 26 26; Fax: +45 35 28 26 06.
bacteria. Useful stains are for instance phenol acetic aniline blue [2] or £uorochromes like acridine orange [3]. Although important information has been obtained by these techniques it is often di¤cult to obtain high quality images due to the complex environment and the small size of the bacteria. Electron microscopy can, on the other hand, provide images of very high resolution. In a series of classical recordings Foster and Rovira [4,5] used transmission electron microscopy (TEM) to study the root-soil interface. While these TEM recordings only show
0168-6496 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 1 6 8 - 6 4 9 6 ( 9 7 ) 0 0 0 3 7 - 8
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very small areas, scanning electron microscopy (SEM) can record larger ¢elds [6]. Both methods, however, su¡er from the rather harsh treatment of the specimen during preparation. Hence, dehydration procedures may seriously distort the structure of the slime matrix (mucilage) around the root [7]. Immuno£uorescence microscopy of selected bacteria using LPS-targeted antibodies coupled to £uorochromes such as £uorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC) remains a key method for strain-speci¢c detection of individual cells in autecological studies. The £uorescent antibody approach has been used to determine the distribution of selected bacteria extracted from rhizosphere environments [8,9]. However, problems with auto£uorescence and non-speci¢c binding of antibody conjugates have been encountered during in situ studies of bacteria directly associated with root tissue [10]. Recently, the development of confocal laser scanning microscopy (CLSM) has signi¢cantly reduced some of these problems. Being a light microscope, this instrument eliminates the dehydration problems associated with preparation for SEM and TEM. Furthermore, the resolution is generally higher and the out-of-focus blur is much reduced compared to conventional £uorescence microscopy. Finally, it is possible to make three-dimensional reconstructions of the recordings and problems with auto£uorescence are much reduced [11]. In this study we have used immuno£uorescence detection by CLSM to study the early colonization of barley roots by Pseudomonas £uorescens strains. Bacterial colonization of the emerging roots immediately after germination may be of crucial importance for the establishment and later development of plants. For instance, fungal pathogens often attack at this time but are inhibited by colonizing bacterial antagonists [12]. In addition, some bacteria colonizing at an early stage directly promote the development of plants [13]. The two strains used were strain DF57 which is antagonistic against the plant pathogen Pythium ultimum, and strain Ag1 without antagonism. The two strains show comparable, but not completely identical Biolog GN substrate utilization patterns. The purpose of this study was to determine the in situ localization pattern for the two strains on developing barley roots to obtain a better understanding of the early colonization process. Further-
more, we studied the simultaneous colonization by the two strains to investigate if mutual interactions would a¡ect their distribution on the root surface. 2. Materials and methods
2.1. Bacterial strains and culture medium P. £uorescens DF57 is a rhizosphere isolate [14] while P. £uorescens Ag1 was originally isolated from bulk soil. Biolog GN ¢ngerprints of the two strains showed that they could utilize 60 and 59 of 95 carbon substrates, respectively; 55 of the substrates were utilized by both strains (cut-o¡ value was set at 3 times the water control). Hence, the niche overlap index (NOI) as de¢ned by Wilson and Lindow [15] was approximately 0.92 (number of carbon sources utilized by both strains as a proportion of number of carbon sources utilized by one strain). At standard conditions the bacteria were grown aerobically in 25 ml Luria broth (LB: 1% Bacto tryptone, 0.5% yeast extract, 1% NaCl and 0.4% glucose) on a rotary shaker (120 rpm), using overnight incubation at room temperature. 2.2. Production of antisera
Antibodies against P. £uorescens DF57 (rabbit anti-DF57) were made by immunizing rabbits (strain Ssc:CPH) subcutaneously with heat-inactivated (100³C for 2 h) cells of DF57 mixed (1:1) with Freund's incomplete adjuvant (State Serum Institute, Denmark). Immunization was done by the State Serum Institute, Denmark according to Harboe and Ingild [16]. NaN3 (15 mM) was added to antisera as a preservative. Speci¢city of the antiserum was determined by dot immunobinding analysis using the following strains: P. £uorescens DSM 50090T , ON5, ON13, ON24, ON25, DF8, DF18, DF23, DF57, DF61, MM5 and MM6, P. putida DSM 291T, ON13, ON31, ON35, DF13, DF14, DF42 and DF45, P. chlororaphis DSM 50083T , P. syringae ATCC 19310T , P. stutzeri DSM 5190T , P. aeruginosa DSM 50071T, Alcaligenes eutrophus DSM 4058, Escherichia coli DSM 498, Bacillus subtilis DSM 402,
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A27, and Vibrio sp. NCIMB 1982 [17]. Weak cross reactions were removed by absorption as described by Nybroe et al. [18] and strain speci¢city assured by repeating the speci¢city test. The immunoglobulin (Ig) G+M fraction was subsequently isolated by ammonium sulfate precipitation [16]. The speci¢city and application of rabbit antibodies to P. £uorescens strain Ag1 (rabbit anti-Ag1) have previously been published [9,18]. Mouse antibodies to Ag1 (mouse anti-Ag1) were made by immunization of mice (strain Ssc:CF1) with heat-inactivated cells as described above for rabbit antiDF57.
Shewanella putrefaciens
2.3. Inoculation and growth of barley plants
The procedures for inoculation of seeds and preparation of plant systems for root colonization studies have been described in detail in Kragelund and Nybroe [9]. In brief, the barley seeds (var. Digger) were surface-sterilized, pre-germinated on moist ¢lter paper for 24 h, and inoculated for 30 min in bacterial suspension. The cell densities for DF57 and Ag1 in the suspensions were adjusted to a ¢nal density of approximately 108 cfu seed31 . For plants to be harvested already after 1 day, the seeds were planted in plastic beakers (diameter: 10 cm) at approximately 1 cm intervals. For plants to be harvested after 7 days, the seeds were placed in 50 ml plastic tubes (1 seed in each tube) with autoclaved soil (sandy loam, 15% water) and incubated at 20³C (12 h dark/12 h light). 2.4. Preparation and immunostaining of roots
Plants were removed from the soil after 1 day or 7 days. The roots were washed gently in 25 mM sodium phosphate bu¡er, 125 mM NaCl, pH 7.4 (PBS) to remove excessive soil, and placed in PBS containing 2% formaldehyde for at least 2 h at room temperature. One-day-old roots were 2^10 mm long and almost devoid of adhering soil, except for a cap of mucilage with adhering soil particles attached to the tip. A total of 5 of these roots were cut o¡ and stained together after the coleorhiza was removed. By comparison, 7-day-old roots were approximately 70 mm long and often contained adhering soil. These
355
roots were cut into 10 equal pieces which were stained separately. Incubation with antibody was performed in a ¢ltration manifold (14 C-Centralen, HÖrsholm, Denmark), as described by Kragelund and Nybroe [9]. Secondary antibodies (Dako, Denmark) were swine anti-rabbit for rabbit anti-DF57/rabbit anti-Ag1 and goat anti-mouse for mouse anti-Ag1, respectively. The secondary antibodies were conjugated to either FITC or TRITC. The TRITC conjugate (for rabbit anti-DF57) was used only when double-staining was performed. The root pieces were adapted to the mounting medium [19] by sequential exposure to 25%, 50% and 75% glycerol. This treatment did not seem to a¡ect the general morphology of the roots as judged from bright ¢eld microscopy. Next, they were mounted between a slide and a coverslip using modelling wax to ensure that the coverslip was placed just on top of the root specimen without squeezing them. 2.5. Confocal laser scanning microscopy
A confocal laser scanning microscope (TCS4d, Leica Laser Technik GmbH, Heidelberg, Germany) was used for detection of the bacteria on the roots. The instrument was equipped with two photomultipliers for £uorescence recordings with the following objectives: 100U plan apo/1.40^0.70 oil, 40U plan £uotar/1.00^0.50 oil, 25U plan £uotar/0.75 oil, 16U plan £uotar/0.50 IMM, and 10U plan £uotar/0.30. In addition to the £uorescence light, the microscope could simultaneously record the light transmitted through the specimen by a third photomultiplier. Recordings with the CLSM were used for in situ determination of bacterial numbers on the 1-dayold roots. The vertical distance between images in the z-plane was 1 Wm, using the 40U objective. Following the initial storing in the computer RAM the images were transferred to a permanent storage on CD-ROM using a Yamaha CDR 102 recorder. Image analysis was performed with the software of the TCS4d. The purpose was to extract the information from a stack of images representing a standard recording into a single image. In most cases this was accomplished by the simulated £uorescence process (SFP; Leica TCS4d manual).
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356
Fig. 1. Colonization of 1-day-old barley roots by
Pseudomonas £uorescens
DF57. A : Transmission image of whole root after preparation
and staining by £uorescent antibody technique. B^D : Fluorescence images by confocal laser scanning microscopy (CLSM) from the locations indicated in A. Mucigel covers the root tip and substantial bacterial colonization can be seen in the area immediately behind the root tip.
situ detection of bacteria on plant roots is the poten-
3. Results and discussion
tial distortion of the specimen during preparation for
3.1. Advantages of CLSM for studies of bacterial root colonization
microscopy. In our experiments, the soil was only loosely attached to the 1-day-old roots and was removed immediately, even when the roots were cau-
By immuno£uorescence staining and three-dimen-
tiously placed in the ¢xative. In contrast, the muci-
sional recordings with CLSM it was possible to de-
lage layer around the 7-day-old roots retained a
termine the localization of two
P. £uorescens
strains
higher amount of soil particles. Since the cap of mu-
at the root surface of barley plants with high reso-
cilage associated with the tip of 1-day-old roots was
lution and speci¢city. A weak auto£uorescence from
still present after preparation, we anticipated that the
the root epidermal cells was always recorded, but did
present protocol only gave a low risk of removing
not seriously a¡ect detection of the bacteria ; by con-
the bacterial population from the rhizoplane.
trast, auto£uorescence made it almost impossible to distinguish the bacteria at the root tip by conven-
3.2. Colonization of 1-day-old barley roots
tional microscopy (data not shown). The auto£uorescence actually turned out to be an advantage for
Fig. 1 shows the distribution of
P. £uorescens
registrations by CLSM as the root cells could be
DF57 on a barley root after 1 day in the soil. As
visualized together with the root-colonizing bacteria.
seen for most roots, this one contained a large num-
Schloter et al. [20] used CLSM to study antibody-
ber of bacteria in the mucilage around the tip. Along
labelled
Azospirillum
signi¢cantly
less
on wheat roots and reported
auto£uorescence
when
bacteria
the root, bacteria tended to be more abundant near the root tip, as observed by rapid monitoring using
were labelled with TRITC than with FITC. We did
conventional
not experience this di¡erence, and detected auto-
shown). To quantify this in detail, recordings with
£uorescence at both ¢lter settings.
the CLSM were made for 15 other roots (4^10 mm
One di¤culty almost invariably associated with in
£uorescence
microscopy
(data
not
long) collected after 1 day. The cell counts obtained
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M. Hansen et al. / FEMS Microbiology Ecology 23 (1997) 353^360
Fig. 2. Fluorescence images by CLSM of
Pseudomonas £uorescens
357
DF57 colonizing the crevices between epithelial cells on the root surface
(rhizoplane) of 1-day-old barley roots. A : Horizontal image showing two strings of bacterial cells occurring along the epithelial cells. B : Vertical image perpendicular to horizontal image at the stippled line. A bacterial cell is seen tightly associated to the epidermal cell in a deep crevice (arrow).
from the root tip (approximately one-quarter of root
in Fig. 2B, where one of the bacteria occurring in a
length, measured from tip) and root base (approxi-
string (Fig. 2A) is located in a deep crevice (arrow).
mately three-quarters of root length, measured from
Such a close association of bacterial cells to the root
tip) environments were 463 þ 80 and 70 þ 15 cells 2 250 m , respectively. Root hairs were developed
surface strongly indicates a tight surface binding of
W 3
on the 1-day-old roots, but were not intensively colonized by
P. £uorescens
DF57 at this time.
the DF57 cells. The distribution pattern on 1-day-old roots indicated a rapid colonization of the rhizoplane, in par-
Apart from the root tip, the 1-day-old plant root
ticular near the root tip. The source of bacteria for
was not intensively covered by mucilage and a large
the colonization was likely to be the mucigel or
proportion of the bacteria was directly associated
sloughed-o¡ epidermal cells at the root apex. If
with the root surface (rhizoplane). Fig. 1 shows
this model of colonization events is valid, it antici-
that after only 1 day of incubation, a signi¢cant
pates active migration by the bacteria from a source
proportion of the bacterial population occurred in
point in the mucigel at the root apex towards the
strings of closely associated cells on the root surface ;
epidermal cells further back on the root. The neces-
furthermore, these strings of bacteria were clearly
sity for chemotactic sensing and active migration in
associated
Pseudomonas
with
the
intercellular
crevices
between
and other bacteria for successful root
neighbouring root epidermal cells. In a few instances
colonization has been subject for debate in the liter-
the auto£uorescence of the epidermal root cells was
ature
quite strong and their cell outlines and the crevices
likely to be attracted by exudate released in the
became clearly visible. In such cases, the recordings
root area immediately behind the apex [1]. In the
indicated that
P. £uorescens
[21^23].
The
root-colonizing
bacteria
were
DF57 cells occurring in
present experiments a role for chemotaxis is further
the strings were closely attached to the root epider-
supported by the predominant location of bacterial
mal cells. This is most clearly shown in the vertical
cells along the cell borders of the root epidermis,
scan of the outer 1^2 layers of epidermal cells shown
where exudation is assumed to be high [1].
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358
Fig. 3. Transmission and £uorescence images by CLSM of
Pseudomonas £uorescens
DF57 colonizing 7-day-old barley roots. Transmission
image A and corresponding £uorescence image B show bacterial cells around the whole cell line of root epidermis. Transmission image C and corresponding £uorescence image D (including images E and F, which correspond to the areas indicated on image C) show a dense rhizoplane population of bacteria assembled in strings in the crevices between root epidermal cells. Dividing cells can be seen in images E and F (arrows).
could follow the whole outline of an epidermal cell
3.3. Colonization of 7-day-old barley roots
(Fig. 3A,B). As shown in Fig. 3C,D, several strings could occur together in a patchy distribution along
The 7-day-old roots were typically covered by a
the root, but the strings never covered a major part
layer of mucilage, which was absent or less devel-
of the root surface as was sometimes observed for
oped on the 1-day-old roots. Within the mucilage
the 1-day-old roots. Cell divisions could be docu-
layer, a population of bacterial cells was found along
mented among the remaining string-forming bacteria
the entire length of the root. This population con-
(Fig. 3E,F, arrows), which indicated that the rhizo-
sisted of single cells or small aggregates of cells,
plane population was still developing after 7 days. In
which were clearly not in tight association with the
further contrast to the pattern observed on the 1-
root epidermal cells (data not shown).
day-old
Fig.
3
shows,
P. £uorescens
however,
that
a
population
roots,
the
rhizoplane-associated
bacteria
of
never appeared at elevated densities near the root
DF57 colonizing the rhizoplane was
tip, and the main roots on 7-day-old plants never
maintained after 7 days of root development. At
showed bacterial strings closer than 5 mm from the
this time, single cells or strings of cells could still
root tip ; it was noticed, however, that small lateral
be found between the root epidermal cells. Usually
roots of the older plants did show strings of bacteria
the bacteria occurring in strings were localized along
close to the tip (data not shown). Finally, root hairs
the longitudinal cell borders, but occasionally they
were well developed on the 7-day-old plants, but
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M. Hansen et al. / FEMS Microbiology Ecology 23 (1997) 353^360
Fig. 4. Fluorescence images by CLSM of
Pseudomonas £uorescens
359
strains DF57 (red) and Ag1 (green) colonizing 1-day-old barley roots.
Images were recorded in (A) mucilage layer around the root tip, (B) rhizoplane, supporting string formation of bacterial population between epidermal cells, and (C) root hair in posterior part of root.
were generally still colonized rather weakly and with-
rhizoplane of the main root (Fig. 4B) ; (3) single cells
out any discernable pattern (data not shown).
on the root hairs (Fig. 4C). In these locations we both observed areas where DF57 was most abun-
3.4. Co-inoculation of Pseudomonas £uorescens strains DF57 and Ag1
dant, areas where Ag1 dominated, and areas where the two strains were equally represented. Consequently,
A series of inoculations with
P. £uorescens
based
on
several
co-inoculation
experi-
strain
ments, the root colonization by strain DF57 did
Ag1 showed patterns of root colonization, which
not seem to be a¡ected by the presence of strain
were very similar to those recorded for strain DF57
Ag1, or vice versa.
(data not shown). The two strains had a high ecological similarity as determined by substrate utilization patterns, and we anticipated that co-existence
Acknowledgments
was inversely related to ecological similarity as has been shown for bacteria in the phyllosphere [15].
This work was supported by the Danish Biotech-
Therefore, if early root colonization comprises sev-
nology Program, the Danish Technical Science Re-
eral phases, which determine bacterial localizations
search Council (Grant no. 16-5232) and The Danish
in time and space as outlined above, it appeared
Veterinary
possible that DF57 and Ag1, competing evenly for
(Grants 13-4898 and 9313839). Thanks are due to
substrates, could co-exist through temporal and spa-
Lene Nielsen for excellent technical assistance with
tial separation.
immuno-staining of root specimens.
and
Agricultural
Research
Council
By conjugating the rabbit anti-DF57 and mouse anti-Ag1 antibodies to red-£uorescing TRITC and green-£uorescing FITC stains, respectively, it was possible to record the two bacteria simultaneously. Fig.
4
shows
recordings
from
such
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Early colonization of barley roots by Pseudomonas £uorescens studied by immuno£uorescence technique and confocal laser scanning microscopy Michael Hansen, Lene Kragelund, Ole Nybroe, Jan SÖrensen * Section of Genetics and Microbiology, Department of Ecology and Molecular Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C (Copenhagen), Denmark Received 29 October 1996; revised 24 April 1997; accepted 6 May 1997
Abstract
Highly specific polyclonal antibodies against two Pseudomonas fluorescens strains (DF57 and Ag1), which differed by approximately 10% of their utilizable substrates as tested in Biolog GN plates, were used for in situ labelling of bacterial cells colonizing barley roots grown in sterile soil. By using a confocal laser scanning microscope single bacteria of both strains could be detected on the roots. Seed-inoculated bacteria rapidly colonized the root surface (rhizoplane) by active migration; after 1 day the anterior part of the root was densely covered by bacteria occupying the crevices between epidermal root cells. As the roots became longer, this bacterial population in the rhizoplane formed long strings of closely associated cells. After 7 days, however, the rhizoplane population of string-forming cells was partially detached developing a patchy distribution along the root; a separate population of cells localized in the slime matrix (mucigel) surrounding the root was well developed at this time. Using two different fluorochromes attached to the antibodies, the two strains could be detected simultaneously in coinoculation experiments. The recordings, however, gave no indications of competition between the two strains during root colonization. Keywords : Root colonization; Pseudomonas £uorescens ; Immuno£uorescence; Confocal laser scanning microscopy
1. Introduction
In situ localizations of bacteria in the rhizosphere have been studied for several decades by using a number of di¡erent microscopic techniques. Light microscopy studies include direct observation of fresh material by phase contrast or interference light microscopy [1] or observations after staining of the * Corresponding author. Tel.: +45 35 28 26 26; Fax: +45 35 28 26 06.
bacteria. Useful stains are for instance phenol acetic aniline blue [2] or £uorochromes like acridine orange [3]. Although important information has been obtained by these techniques it is often di¤cult to obtain high quality images due to the complex environment and the small size of the bacteria. Electron microscopy can, on the other hand, provide images of very high resolution. In a series of classical recordings Foster and Rovira [4,5] used transmission electron microscopy (TEM) to study the root-soil interface. While these TEM recordings only show
0168-6496 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 1 6 8 - 6 4 9 6 ( 9 7 ) 0 0 0 3 7 - 8
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very small areas, scanning electron microscopy (SEM) can record larger ¢elds [6]. Both methods, however, su¡er from the rather harsh treatment of the specimen during preparation. Hence, dehydration procedures may seriously distort the structure of the slime matrix (mucilage) around the root [7]. Immuno£uorescence microscopy of selected bacteria using LPS-targeted antibodies coupled to £uorochromes such as £uorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC) remains a key method for strain-speci¢c detection of individual cells in autecological studies. The £uorescent antibody approach has been used to determine the distribution of selected bacteria extracted from rhizosphere environments [8,9]. However, problems with auto£uorescence and non-speci¢c binding of antibody conjugates have been encountered during in situ studies of bacteria directly associated with root tissue [10]. Recently, the development of confocal laser scanning microscopy (CLSM) has signi¢cantly reduced some of these problems. Being a light microscope, this instrument eliminates the dehydration problems associated with preparation for SEM and TEM. Furthermore, the resolution is generally higher and the out-of-focus blur is much reduced compared to conventional £uorescence microscopy. Finally, it is possible to make three-dimensional reconstructions of the recordings and problems with auto£uorescence are much reduced [11]. In this study we have used immuno£uorescence detection by CLSM to study the early colonization of barley roots by Pseudomonas £uorescens strains. Bacterial colonization of the emerging roots immediately after germination may be of crucial importance for the establishment and later development of plants. For instance, fungal pathogens often attack at this time but are inhibited by colonizing bacterial antagonists [12]. In addition, some bacteria colonizing at an early stage directly promote the development of plants [13]. The two strains used were strain DF57 which is antagonistic against the plant pathogen Pythium ultimum, and strain Ag1 without antagonism. The two strains show comparable, but not completely identical Biolog GN substrate utilization patterns. The purpose of this study was to determine the in situ localization pattern for the two strains on developing barley roots to obtain a better understanding of the early colonization process. Further-
more, we studied the simultaneous colonization by the two strains to investigate if mutual interactions would a¡ect their distribution on the root surface. 2. Materials and methods
2.1. Bacterial strains and culture medium P. £uorescens DF57 is a rhizosphere isolate [14] while P. £uorescens Ag1 was originally isolated from bulk soil. Biolog GN ¢ngerprints of the two strains showed that they could utilize 60 and 59 of 95 carbon substrates, respectively; 55 of the substrates were utilized by both strains (cut-o¡ value was set at 3 times the water control). Hence, the niche overlap index (NOI) as de¢ned by Wilson and Lindow [15] was approximately 0.92 (number of carbon sources utilized by both strains as a proportion of number of carbon sources utilized by one strain). At standard conditions the bacteria were grown aerobically in 25 ml Luria broth (LB: 1% Bacto tryptone, 0.5% yeast extract, 1% NaCl and 0.4% glucose) on a rotary shaker (120 rpm), using overnight incubation at room temperature. 2.2. Production of antisera
Antibodies against P. £uorescens DF57 (rabbit anti-DF57) were made by immunizing rabbits (strain Ssc:CPH) subcutaneously with heat-inactivated (100³C for 2 h) cells of DF57 mixed (1:1) with Freund's incomplete adjuvant (State Serum Institute, Denmark). Immunization was done by the State Serum Institute, Denmark according to Harboe and Ingild [16]. NaN3 (15 mM) was added to antisera as a preservative. Speci¢city of the antiserum was determined by dot immunobinding analysis using the following strains: P. £uorescens DSM 50090T , ON5, ON13, ON24, ON25, DF8, DF18, DF23, DF57, DF61, MM5 and MM6, P. putida DSM 291T, ON13, ON31, ON35, DF13, DF14, DF42 and DF45, P. chlororaphis DSM 50083T , P. syringae ATCC 19310T , P. stutzeri DSM 5190T , P. aeruginosa DSM 50071T, Alcaligenes eutrophus DSM 4058, Escherichia coli DSM 498, Bacillus subtilis DSM 402,
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A27, and Vibrio sp. NCIMB 1982 [17]. Weak cross reactions were removed by absorption as described by Nybroe et al. [18] and strain speci¢city assured by repeating the speci¢city test. The immunoglobulin (Ig) G+M fraction was subsequently isolated by ammonium sulfate precipitation [16]. The speci¢city and application of rabbit antibodies to P. £uorescens strain Ag1 (rabbit anti-Ag1) have previously been published [9,18]. Mouse antibodies to Ag1 (mouse anti-Ag1) were made by immunization of mice (strain Ssc:CF1) with heat-inactivated cells as described above for rabbit antiDF57.
Shewanella putrefaciens
2.3. Inoculation and growth of barley plants
The procedures for inoculation of seeds and preparation of plant systems for root colonization studies have been described in detail in Kragelund and Nybroe [9]. In brief, the barley seeds (var. Digger) were surface-sterilized, pre-germinated on moist ¢lter paper for 24 h, and inoculated for 30 min in bacterial suspension. The cell densities for DF57 and Ag1 in the suspensions were adjusted to a ¢nal density of approximately 108 cfu seed31 . For plants to be harvested already after 1 day, the seeds were planted in plastic beakers (diameter: 10 cm) at approximately 1 cm intervals. For plants to be harvested after 7 days, the seeds were placed in 50 ml plastic tubes (1 seed in each tube) with autoclaved soil (sandy loam, 15% water) and incubated at 20³C (12 h dark/12 h light). 2.4. Preparation and immunostaining of roots
Plants were removed from the soil after 1 day or 7 days. The roots were washed gently in 25 mM sodium phosphate bu¡er, 125 mM NaCl, pH 7.4 (PBS) to remove excessive soil, and placed in PBS containing 2% formaldehyde for at least 2 h at room temperature. One-day-old roots were 2^10 mm long and almost devoid of adhering soil, except for a cap of mucilage with adhering soil particles attached to the tip. A total of 5 of these roots were cut o¡ and stained together after the coleorhiza was removed. By comparison, 7-day-old roots were approximately 70 mm long and often contained adhering soil. These
355
roots were cut into 10 equal pieces which were stained separately. Incubation with antibody was performed in a ¢ltration manifold (14 C-Centralen, HÖrsholm, Denmark), as described by Kragelund and Nybroe [9]. Secondary antibodies (Dako, Denmark) were swine anti-rabbit for rabbit anti-DF57/rabbit anti-Ag1 and goat anti-mouse for mouse anti-Ag1, respectively. The secondary antibodies were conjugated to either FITC or TRITC. The TRITC conjugate (for rabbit anti-DF57) was used only when double-staining was performed. The root pieces were adapted to the mounting medium [19] by sequential exposure to 25%, 50% and 75% glycerol. This treatment did not seem to a¡ect the general morphology of the roots as judged from bright ¢eld microscopy. Next, they were mounted between a slide and a coverslip using modelling wax to ensure that the coverslip was placed just on top of the root specimen without squeezing them. 2.5. Confocal laser scanning microscopy
A confocal laser scanning microscope (TCS4d, Leica Laser Technik GmbH, Heidelberg, Germany) was used for detection of the bacteria on the roots. The instrument was equipped with two photomultipliers for £uorescence recordings with the following objectives: 100U plan apo/1.40^0.70 oil, 40U plan £uotar/1.00^0.50 oil, 25U plan £uotar/0.75 oil, 16U plan £uotar/0.50 IMM, and 10U plan £uotar/0.30. In addition to the £uorescence light, the microscope could simultaneously record the light transmitted through the specimen by a third photomultiplier. Recordings with the CLSM were used for in situ determination of bacterial numbers on the 1-dayold roots. The vertical distance between images in the z-plane was 1 Wm, using the 40U objective. Following the initial storing in the computer RAM the images were transferred to a permanent storage on CD-ROM using a Yamaha CDR 102 recorder. Image analysis was performed with the software of the TCS4d. The purpose was to extract the information from a stack of images representing a standard recording into a single image. In most cases this was accomplished by the simulated £uorescence process (SFP; Leica TCS4d manual).
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356
Fig. 1. Colonization of 1-day-old barley roots by
Pseudomonas £uorescens
DF57. A : Transmission image of whole root after preparation
and staining by £uorescent antibody technique. B^D : Fluorescence images by confocal laser scanning microscopy (CLSM) from the locations indicated in A. Mucigel covers the root tip and substantial bacterial colonization can be seen in the area immediately behind the root tip.
situ detection of bacteria on plant roots is the poten-
3. Results and discussion
tial distortion of the specimen during preparation for
3.1. Advantages of CLSM for studies of bacterial root colonization
microscopy. In our experiments, the soil was only loosely attached to the 1-day-old roots and was removed immediately, even when the roots were cau-
By immuno£uorescence staining and three-dimen-
tiously placed in the ¢xative. In contrast, the muci-
sional recordings with CLSM it was possible to de-
lage layer around the 7-day-old roots retained a
termine the localization of two
P. £uorescens
strains
higher amount of soil particles. Since the cap of mu-
at the root surface of barley plants with high reso-
cilage associated with the tip of 1-day-old roots was
lution and speci¢city. A weak auto£uorescence from
still present after preparation, we anticipated that the
the root epidermal cells was always recorded, but did
present protocol only gave a low risk of removing
not seriously a¡ect detection of the bacteria ; by con-
the bacterial population from the rhizoplane.
trast, auto£uorescence made it almost impossible to distinguish the bacteria at the root tip by conven-
3.2. Colonization of 1-day-old barley roots
tional microscopy (data not shown). The auto£uorescence actually turned out to be an advantage for
Fig. 1 shows the distribution of
P. £uorescens
registrations by CLSM as the root cells could be
DF57 on a barley root after 1 day in the soil. As
visualized together with the root-colonizing bacteria.
seen for most roots, this one contained a large num-
Schloter et al. [20] used CLSM to study antibody-
ber of bacteria in the mucilage around the tip. Along
labelled
Azospirillum
signi¢cantly
less
on wheat roots and reported
auto£uorescence
when
bacteria
the root, bacteria tended to be more abundant near the root tip, as observed by rapid monitoring using
were labelled with TRITC than with FITC. We did
conventional
not experience this di¡erence, and detected auto-
shown). To quantify this in detail, recordings with
£uorescence at both ¢lter settings.
the CLSM were made for 15 other roots (4^10 mm
One di¤culty almost invariably associated with in
£uorescence
microscopy
(data
not
long) collected after 1 day. The cell counts obtained
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M. Hansen et al. / FEMS Microbiology Ecology 23 (1997) 353^360
Fig. 2. Fluorescence images by CLSM of
Pseudomonas £uorescens
357
DF57 colonizing the crevices between epithelial cells on the root surface
(rhizoplane) of 1-day-old barley roots. A : Horizontal image showing two strings of bacterial cells occurring along the epithelial cells. B : Vertical image perpendicular to horizontal image at the stippled line. A bacterial cell is seen tightly associated to the epidermal cell in a deep crevice (arrow).
from the root tip (approximately one-quarter of root
in Fig. 2B, where one of the bacteria occurring in a
length, measured from tip) and root base (approxi-
string (Fig. 2A) is located in a deep crevice (arrow).
mately three-quarters of root length, measured from
Such a close association of bacterial cells to the root
tip) environments were 463 þ 80 and 70 þ 15 cells 2 250 m , respectively. Root hairs were developed
surface strongly indicates a tight surface binding of
W 3
on the 1-day-old roots, but were not intensively colonized by
P. £uorescens
DF57 at this time.
the DF57 cells. The distribution pattern on 1-day-old roots indicated a rapid colonization of the rhizoplane, in par-
Apart from the root tip, the 1-day-old plant root
ticular near the root tip. The source of bacteria for
was not intensively covered by mucilage and a large
the colonization was likely to be the mucigel or
proportion of the bacteria was directly associated
sloughed-o¡ epidermal cells at the root apex. If
with the root surface (rhizoplane). Fig. 1 shows
this model of colonization events is valid, it antici-
that after only 1 day of incubation, a signi¢cant
pates active migration by the bacteria from a source
proportion of the bacterial population occurred in
point in the mucigel at the root apex towards the
strings of closely associated cells on the root surface ;
epidermal cells further back on the root. The neces-
furthermore, these strings of bacteria were clearly
sity for chemotactic sensing and active migration in
associated
Pseudomonas
with
the
intercellular
crevices
between
and other bacteria for successful root
neighbouring root epidermal cells. In a few instances
colonization has been subject for debate in the liter-
the auto£uorescence of the epidermal root cells was
ature
quite strong and their cell outlines and the crevices
likely to be attracted by exudate released in the
became clearly visible. In such cases, the recordings
root area immediately behind the apex [1]. In the
indicated that
P. £uorescens
[21^23].
The
root-colonizing
bacteria
were
DF57 cells occurring in
present experiments a role for chemotaxis is further
the strings were closely attached to the root epider-
supported by the predominant location of bacterial
mal cells. This is most clearly shown in the vertical
cells along the cell borders of the root epidermis,
scan of the outer 1^2 layers of epidermal cells shown
where exudation is assumed to be high [1].
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M. Hansen et al. / FEMS Microbiology Ecology 23 (1997) 353^360
358
Fig. 3. Transmission and £uorescence images by CLSM of
Pseudomonas £uorescens
DF57 colonizing 7-day-old barley roots. Transmission
image A and corresponding £uorescence image B show bacterial cells around the whole cell line of root epidermis. Transmission image C and corresponding £uorescence image D (including images E and F, which correspond to the areas indicated on image C) show a dense rhizoplane population of bacteria assembled in strings in the crevices between root epidermal cells. Dividing cells can be seen in images E and F (arrows).
could follow the whole outline of an epidermal cell
3.3. Colonization of 7-day-old barley roots
(Fig. 3A,B). As shown in Fig. 3C,D, several strings could occur together in a patchy distribution along
The 7-day-old roots were typically covered by a
the root, but the strings never covered a major part
layer of mucilage, which was absent or less devel-
of the root surface as was sometimes observed for
oped on the 1-day-old roots. Within the mucilage
the 1-day-old roots. Cell divisions could be docu-
layer, a population of bacterial cells was found along
mented among the remaining string-forming bacteria
the entire length of the root. This population con-
(Fig. 3E,F, arrows), which indicated that the rhizo-
sisted of single cells or small aggregates of cells,
plane population was still developing after 7 days. In
which were clearly not in tight association with the
further contrast to the pattern observed on the 1-
root epidermal cells (data not shown).
day-old
Fig.
3
shows,
P. £uorescens
however,
that
a
population
roots,
the
rhizoplane-associated
bacteria
of
never appeared at elevated densities near the root
DF57 colonizing the rhizoplane was
tip, and the main roots on 7-day-old plants never
maintained after 7 days of root development. At
showed bacterial strings closer than 5 mm from the
this time, single cells or strings of cells could still
root tip ; it was noticed, however, that small lateral
be found between the root epidermal cells. Usually
roots of the older plants did show strings of bacteria
the bacteria occurring in strings were localized along
close to the tip (data not shown). Finally, root hairs
the longitudinal cell borders, but occasionally they
were well developed on the 7-day-old plants, but
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M. Hansen et al. / FEMS Microbiology Ecology 23 (1997) 353^360
Fig. 4. Fluorescence images by CLSM of
Pseudomonas £uorescens
359
strains DF57 (red) and Ag1 (green) colonizing 1-day-old barley roots.
Images were recorded in (A) mucilage layer around the root tip, (B) rhizoplane, supporting string formation of bacterial population between epidermal cells, and (C) root hair in posterior part of root.
were generally still colonized rather weakly and with-
rhizoplane of the main root (Fig. 4B) ; (3) single cells
out any discernable pattern (data not shown).
on the root hairs (Fig. 4C). In these locations we both observed areas where DF57 was most abun-
3.4. Co-inoculation of Pseudomonas £uorescens strains DF57 and Ag1
dant, areas where Ag1 dominated, and areas where the two strains were equally represented. Consequently,
A series of inoculations with
P. £uorescens
based
on
several
co-inoculation
experi-
strain
ments, the root colonization by strain DF57 did
Ag1 showed patterns of root colonization, which
not seem to be a¡ected by the presence of strain
were very similar to those recorded for strain DF57
Ag1, or vice versa.
(data not shown). The two strains had a high ecological similarity as determined by substrate utilization patterns, and we anticipated that co-existence
Acknowledgments
was inversely related to ecological similarity as has been shown for bacteria in the phyllosphere [15].
This work was supported by the Danish Biotech-
Therefore, if early root colonization comprises sev-
nology Program, the Danish Technical Science Re-
eral phases, which determine bacterial localizations
search Council (Grant no. 16-5232) and The Danish
in time and space as outlined above, it appeared
Veterinary
possible that DF57 and Ag1, competing evenly for
(Grants 13-4898 and 9313839). Thanks are due to
substrates, could co-exist through temporal and spa-
Lene Nielsen for excellent technical assistance with
tial separation.
immuno-staining of root specimens.
and
Agricultural
Research
Council
By conjugating the rabbit anti-DF57 and mouse anti-Ag1 antibodies to red-£uorescing TRITC and green-£uorescing FITC stains, respectively, it was possible to record the two bacteria simultaneously. Fig.
4
shows
recordings
from
such
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