FEMS Microbiology Ecology 47 (2004) 319^326
www.fems-microbiology.org
Plant colonization by pink-pigmented facultative methylotrophic
bacteria (PPFMs)
Zahra S. Omer , Riccardo Tombolini, Berndt Gerhardson
Plant Pathology and Biocontrol Unit, Box 7035, S-750 07 Uppsala, Sweden
Received 11 August 2003 ; received in revised form 5 November 2003; accepted 17 November 2003
First published online 26 January 2004
Abstract
Bacteria belonging to the genus Methylobacterium are characterized by being able to rely on methanol as a sole carbon and energy
source and by presenting a more or less intense pink reddish pigmentation. These bacteria, also referred to as pink-pigmented
methylotrophic bacteria (PPFMs), are common inhabitants of the phyllosphere and are found in many other environmental samples.
Since they grow slowly they are often overlooked and their impact on phyllosphere microbial communities and on the plants harboring
them is not well studied nor has their ecology been elucidated. In a survey of PPFM colonization in three different agricultural sites,
PPFM populations were identified on both red clover and winter wheat, but red clover was more consistently colonized. Isolations from
collected leaves showed PPFM populations to decrease from spring towards summer, but they increased again towards the end of the
cropping season. Isolates from red clover readily colonized winter wheat leaves and vice versa in greenhouse experiments, but population
sizes were dependent on the application procedure. Tested isolates had also good potential to colonize the rhizosphere, especially after
seed inoculations. Confocal scanning laser microscopy showed gfp-tagged isolates to colonize the surface of clover leaves by forming large
aggregates.
6 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords : Methylobacterium ; Rhizosphere ; Phyllosphere; Colonization ; Trifolium pratense; Triticum aestivum
1. Introduction
Bacteria belonging to the genus Methylobacterium,
known as pink-pigmented facultative methylotrophic bacteria (PPFMs), are ubiquitous in nature and have been
detected in soil, dust, freshwater, lake sediments, on leaf
surfaces and nodules, in rice grains, air, hospital environments, as well as on other solid surfaces [1,2]. They are
aerobic, Gram-negative bacteria and although they are
able to grow on a wide range of multi-carbon substrates,
they are characterized by the capability to grow on onecarbon compounds such as formate, formaldehyde or
methanol as the sole carbon and energy source. They are
thus easily isolated on a methanol-based mineral medium
[1,2].
The frequently reported isolations of PPFMs from plant
material, in particular from leaf surfaces [3], and the
proved association with more than 70 plant species [2,4]
* Corresponding author. Tel.: +46 (18) 671605; Fax: +46 (18) 671690.
E-mail address : [email protected] (Z.S. Omer).
make them interesting as potential agents a¡ecting plant
growth and/or suppressing disease. However, there are so
far only few reports focusing on these aspects. Certain
isolates are known to produce auxins [5], cytokinins [6,7]
and vitamin B12 [8]. Interactions with the plant nitrogen
metabolism mediated by bacterial urease and the possible
role of this in seed germination physiology have also been
described [4]. Bacteria of the genus Methylobacterium
were, furthermore, found to nodulate legumes of the genus
Crotalaria [9] indicating strong plant^bacteria interactions.
As for the PPFM populations on crop plants and their
dynamics, available data are contradictory and fragmentary. PPFM populations of white clover plants have been
described on a two-sampling base and higher levels of
PPFMs associated with leaves were detected during the
summer months in comparison with the population detected during early spring and late fall [2]. In another
limited survey white clover samples were collected during
1.5 months and no population changes occurred during
this period [3]. On the other hand Hirano et al. [10] in a
1.5-month survey on snap bean found population variations during the considered sampling period.
0168-6496 / 04 / $22.00 6 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/S0168-6496(04)00003-0
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Z.S. Omer et al. / FEMS Microbiology Ecology 47 (2004) 319^326
In a study on colonization on maize by PPFMs, it was
found that leaves are not colonized after seed bacterization
and/or after soil application of a PPFM strain, but only
after direct application on the phyllosphere, and it was
suggested that natural leaf colonization occurs via air
transfer of soil particles [11]. However, Holland and Polacco [12], from indirect evidence, suggested that leaf-inhabiting PPFMs are probably the descendants of seedborne bacteria rather than bacteria from air, soil, or water,
or derived from other plants.
In the present paper we address the populations of these
microorganisms on crop plants in northern Europe, the
variability of PPFM colonization during the growing season and the e¡ect of crop plant species. In order to get
insight into the PPFM^plant system and to set up a suitable protocol for the assessment of plant bene¢cial e¡ects
and possible biocontrol, we also report on plant colonization after inoculation of two kanr -gfp-tagged PPFM isolates under greenhouse conditions.
Localization and the pattern of colonization of kanr gfp-tagged strains on inoculated plants were determined
using confocal scanning laser microscopy (CSLM).
2. Materials and methods
2.1. Bacterial strains and cultivation
For maintenance of PPFMs and plating of environmental samples an ammonium mineral salts medium considered to be selective for PPFMs was used [2] (AMS: per
liter, 0.7 g K2 HPO4 , 0.54 g KH2 PO4 , 1 g MgSO4 W7H2 O,
0.2 g CaCl2 W2H2 O, 4 mg FeSO4 W7H2 O, 0.5 g NH4 Cl, 100
Wg ZnSO4 W7H2 O, 30 Wg MnCl2 W4H2 O, 300 Wg H3 BO3 , 200
Wg CoCl2 W6H2 O, 10 Wg CuCl2 W2H2 O, 20 Wg NiCl2 W6H2 O, 60
Wg Na2 MoO4 W2H2 O, 15 g agar technical and 0.5% methanol). For the transformation of PPFM isolates and for
Escherichia coli strain the following culture media were
used : tryptic soy broth (TSB: 15 g tryptic soy broth (Difco) in 1000 ml distilled H2 O); Luria broth (LB: 10 g
tryptone, 5 g yeast extract, 10 g NaCl in 1000 ml distilled
H2 O). Two PPFM isolates chosen for extended greenhouse studies, Mb49 (16S rRNA gene sequence GenBank
accession number : AY248705), and HSC5 (16S rRNA
gene sequence GenBank accession number: AY158812),
were isolated from winter wheat and red clover leaves,
respectively. These strains both produce cytokinin, HSC5
is also able to produce auxin (unpublished results) and
Mb49 has been shown to induce antifungal and antibacterial activity (J. Borowicz, unpublished results).
2.2. Construction of gfp-tagged mutants
Strains Mb49 and HSC5 were tagged with gfp in the
chromosome using the mini-Tn5-based gfp delivery vector
pUTgfp2X [13] and transformed by conjugation according
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to a previously described protocol [14]. Transconjugants
were plated on AMS amended with 50 Wg ml31 kanamycin
(kan) and selected on the basis of £uorescence intensity
and similarity to the corresponding wild-type. In order
to assess the stability of the introduced genetic markers
(kanr and gfp) the mutants were grown on non-selective
liquid medium for 2 days and then inoculated in new
medium 10 times. The last bacterial suspensions were
plated on non-selective solid medium and 100% (300 random colonies checked) of the colonies were kanamycinresistant and green £uorescent. The stability of the
markers was also con¢rmed by a starvation experiment.
Mutant strains were scraped-o¡ from four-days-old agar
plate cultures and were washed two times in sterile water
and kept at 4‡C for 2 weeks. After plating on non-selective
medium 100% of the colonies (300 random colonies
checked) expressed green £uorescent protein (GFP) and
resistance to kanamycin. The mutant strains were compared with the wild-type with respect to colony appearance, on both AMS and rich medium, and growth rate on
AMS. The two mutants did not show any di¡erence from
the corresponding wild-type strains. Colonies on agar
plates showed a weak green £uorescence, but the gfptagged bacteria were readily detected under the epi£uorescence microscope and confocal microscope.
In the experiments where the applied bacteria were
monitored for their colonization ability, selectivity of the
AMS medium was achieved by including kanamycin,
whereas the green £uorescence of representative pink-pigmented colonies was checked by means of an epi£uorescence microscope.
2.3. Assessment of natural PPFM populations on
¢eld-grown plants
The populations of PPFMs were estimated during the
years 2001 and 2002 on winter wheat (Triticum aestivum)
and red clover (Trifolium pratense) collected at three di¡erent sites, Sa«by, Hammarby and Hammarskog, near Uppsala, Sweden. Three samples, approximately 20 m apart,
were collected from a number of plants from each location
for each crop plant and transported separately in plastic
bags to the lab. The fresh weight (FW) of each sample was
recorded before further processing. The leaves were homogenized in 10 ml of sterilized distilled water in sterilized
mortars. Serial dilutions in sterilized distilled water were
plated in triplicate onto AMS by the drop plate method
(detection limit of 103 CFU ml31 g31 ) [15]. We modi¢ed
the procedure slightly by spotting four drops of bacterial
dilution per plate and letting them slide by tilting the
plate, in order to distribute the drop on a strip about
5 cm long. This facilitated the counting. Plates were incubated at 25‡C for 7 days before counting of the colonies.
The CFU values were expressed as log CFU (g FW)31 . In
Table 1 the values of temperature, relative humidity and
precipitation on the sampling days are reported.
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Z.S. Omer et al. / FEMS Microbiology Ecology 47 (2004) 319^326
2.4. Plant colonization by kanr -gfp-tagged PPFMs in
greenhouse experiments
Winter wheat and red clover were used as model plants
for testing the colonization capability of the selected
strains (Mb49: :gfp and HSC5: :gfp). Tagged bacteria
were inoculated onto plants grown in a greenhouse. Population levels were then estimated weekly by plate counting on AMS-Kan.
For seed bacterization, the inoculum was prepared by
scraping o¡ bacterial cells from AMS-Kan agar plate. The
cells were then suspended in distilled water in a test tube,
centrifuged and washed three times before being resuspended in distilled water to a concentration of 109 CFU
ml31 . Three grams of either wheat or red clover seeds were
soaked in the bacterial suspension for 1 h and then directly
sown in 12.5U13.5U7-cm pots containing a peat mix soil.
The soil mix composition was the following: 60% low
humi¢ed peat, 30% high humi¢ed peat and 10% sand,
fertilized with dolomite, lime, macronutrients 12-5-14
(NPK) and micronutrients (FTE 36 0.1 kg m33 ), pH
5.5^6.5. Greenhouse temperature was kept at a 25/20‡C
day/night regime and at approximately 70% relative humidity. Twelve wheat plants were sown per pot and three
of these used for each sampling. About 20 clover plants
were sown in each pot and three to four plants were used
for each sampling.
For spray application on leaves and shoots, the bacterial suspension was prepared as described above. The ¢nal
concentration of the bacterial suspension was 109 CFU
ml31 in water or in a 0.05% solution of an organosilicone/linear alcohol surfactant blend (Slippa1, Interagro
UK, Herts, UK) in water. The shoots of 7-day-old plants
were sprayed with 10 ml bacterial suspension in each pot.
To determine the population levels the roots were excised from the same plants from which leaf samples were
obtained, and the aerial plant parts were cut 5 cm above
soil level to avoid soil contamination. Both root and leaf
samples were homogenized in 10 ml of sterilized distilled
water in sterile mortars. In a second set of experiments
Table 1
Weather parameters on the days of ¢eld sampling obtained from Ultuna
climate station, SLU
Date of
sampling
Year 2001
April 2
May 2
July 2
August 2
Year 2002
March 27
April 16
June 18
June 27
July 9
Temperature
(‡C)
Relative
humidity (%)
321
with spray application, clover and wheat plants were
sprayed 2 weeks after seed germination when red clover
plants had two di¡erent leaves, the cotyledons (leaf A) and
the unifoliate leaf (leaf B). Wheat plants were at the ¢rst
leaf stage with coleoptiles (leaf A) and the ¢rst leaf (leaf
B). Samples were then collected as single leaf samples
for checking the presence of bacteria in newly emerging
leaves.
In all the experiments where the plants were treated with
the two PPFM isolates, no macroscopic di¡erences from
the untreated controls were observed.
2.5. In situ localization of gfp-tagged PPFMs
Approximately 0.5-cm2 leaf pieces were cut o¡ from
clover and wheat leaves sprayed with Mb49 : :gfp 3 days
after bacterial application. The leaf pieces were directly
placed on a microscopy slide and a drop of Vectashield
mounting medium (Vector Laboratories, Burlingame, CA,
USA) was added to the specimen before placement of a
coverslip and visualization by CSLM. A CSLM Leica
model TCS with excitation wavelength of 488 nm (Ar
laser) and 633 nm (HeNe laser) was used. Emission light
was collected in a range of 510^560 nm for GFP and 620^
660 nm for background £uorescence and a U40 objective
was used. For PPFM visualization in the root system,
root pieces were excised from washed roots of 5-day-old
seedlings originating from seeds bacterized with strain
Mb49: :gfp.
2.6. Statistical analysis
The ¢eld sampling data of the PPFM natural populations were analyzed by the General Linear Model, GLM,
SAS package. The model adopted was:
A [log CFU (g FW)31 ] = Y (year) L (location) M
(month) P (plant species). Mean values of di¡erent treatments were compared using Duncan’s multiple range test
at P = 0.05 level.
Data on plant colonization were subjected to one-way
analysis of variance. Treatments were run in six replicates
in the seed bacterization experiment whereas all the treatments in spray experiments were run in triplicate. Data in
¢gures are expressed as mean log CFU (g FW)31 V S.D.
Precipitation
(mm)
3. Results and discussion
7.5
11.6
17.9
14.3
70
54
60
60
0
0
0
0
34.5
1.3
22.2
12.3
19.5
90
81
65
86
73
3.1
0
0.6
10.0
0
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3.1. Natural PPFM population in ¢eld-grown crops
The red clover plants sampled showed overall PPFM
population levels about 2.5 orders of magnitude higher
than those of winter wheat (Table 2). For red clover the
highest PPFM population was recorded at the time of the
¢rst sampling, corresponding to the onset of growth in
March^April (Fig. 1 and Table 2). The bacterial popula-
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Z.S. Omer et al. / FEMS Microbiology Ecology 47 (2004) 319^326
tion level then decreased during the spring and subsequently increased again in the summer until harvesting
time (Fig. 1). The bacterial population level, variance of
the data and trend in population changes throughout the
growing season were comparable in both years (Fig. 1),
and no positive correlations with crop location were detected (Pr = 0.9541).
In the winter wheat the lower PPFM levels (close to the
experimental detection limit of log 3 CFU (g FW)31 ) and
the higher variability among samples made it impossible to
statistically validate the population pattern during the
growing season. However, winter wheat PPFM populations showed a trend similar to red clover, with a higher
bacterial population level at the beginning and at the end
of the growing season (Table 2 and Fig. 1).
The month of the year/plant growing stage factor signi¢cantly in£uenced the PPFM population sizes on the
sampled leaves (Pr = 0.0001 both for clover and wheat).
Using pooled data of both plant species for each year, a
signi¢cant di¡erence in the total bacterial counts could be
recorded between the two years (Pr = 0.0166) and the plant
species tested (Pr = 0.0001).
It is well established that PPFMs are leaf inhabitants
[2,4], but these data have mainly been obtained using
leaf impression methods on methanol-agar media. Limited
data are thus available for their quanti¢cation and population variation on plants. Corpe and Rheem [3] reported
high PPFM levels on white clover in sampling from May
until mid-July, and no changes or trends were recognized.
Our results show, in contrast, a clear trend towards an
increase of bacterial counts on leaves following a signi¢cant decrease in the late spring. A high bacterial population on plant parts closer to the soil has been considered a
general feature [16], and this might be an explanation for
the relatively high level of PPFMs found in both red clover and winter wheat at the ¢rst yearly samplings when
plants were in the seedling stage. Although the perennial
Table 2
GLM, Duncan’s multiple range test for variable log CFU (g FW)31 at
sampling dates during the growing season
Month of sampling
Clover
March
April
May
June (early)
June (late)
July
August
Wheat
April
May
June (early)
June (late)
July
August
Mean log CFU (g FW)31
Duncan grouping
5.83
5.76
2.45
3.18
3.93
3.93
3.82
A
A
C
BC
B
B
B
1.92
0.00
0.3
2.56
2.23
2.67
A
B
B
A
A
A
Data collected in 2001 and 2002 were pooled for each plant species.
FEMSEC 1621 20-2-04
Fig. 1. Population changes of PPFMs in red clover phyllosphere in two
di¡erent years, 2001 (A) and 2002 (B). Error bars are V S.D. *Bacterial
population below the detection limit.
red clover was not sown in the ¢eld surveyed, close contact
with the soil before re-growth might have contributed to
the high bacterial counts in the early spring. The possibility that the changes in CFU levels during the growing
season correspond to variation in culturability (VBNC
state) cannot be ruled out. The possible entrance into
the VBNC state by PPFMs has not been studied yet.
The population increase in late spring/summer indicates
increasingly suitable conditions for Methylobacterium spp.
as the plants grow and the leaves age. Climatic conditions
might also be a positive selective factor. Their resistance to
desiccation, UV light [17], and chlorine treatment, and the
¢nding that some species are resistant to penicillin, chloramphenicol as well as other antibiotics [18], should give
them good adaptation abilities on the leaves during plant
maturation. The reported higher PPFM population levels
during summer periods in temperate geographic regions,
where UV irradiation, temperature and water stresses play
a role, raises the question about PPFM impact on crops in
cold and humid regions. Our results indicate that cold
temperatures do not impair colonization by PPFMs.
The pooling of leaves during our sampling may have
attenuated the variance of the data in the case of clover,
whereas it did not in the same way compensate an intrinsic
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Z.S. Omer et al. / FEMS Microbiology Ecology 47 (2004) 319^326
Fig. 2. Root and leaf colonization of red clover and root colonization
of wheat by two gfp-tagged PPFMs after seed bacterization. Isolate
Mb49 from winter wheat; isolate HSC5 from red clover. c, clover ; w,
wheat. Error bars indicate V S.D.
high leaf-to-leaf variability in wheat. On the other hand,
Hirano and Upper [10] found that PPFMs, unlike other
phyllosphere-inhabiting bacteria, exhibit population levels
derived from single leaf samples similar to the ones derived from bulk samples. It is possible that clover plants
possess a higher carrying capacity than wheat plants for
PPFMs, as our greenhouse experiments seem to con¢rm
(see below, Fig. 3). The reason for this is so far unknown.
The observation that dicotyledonous cell walls contain
more pectin, the source of plant methanol, than the walls
of grasses [19] might explain a lower ¢tness of PPFMs in
wheat plants than in clover, but data showing such connections are still lacking.
323
are part of the natural rhizosphere microbial population,
but presently ongoing isolations from the rhizospheres of
¢eld-grown plants have shown that using selective media
these microorganisms are always present (results not
shown).
Isolates experimentally applied to clover seeds were able
to colonize not only the roots but also the phyllosphere
(Fig. 2). This was, however, not the case for bacteria applied to wheat seeds (Fig. 2), indicating a di¡erence between plants also in this case. Romanovskaya et al. [11]
likewise did not ¢nd PPFMs on maize leaves after soil
inoculation and seed applications. Thus, the capability of
these bacteria to e⁄ciently colonize the roots and the aerial plant parts from a seed or soil source appears to be
plant species-dependent.
3.3. Phyllosphere colonization after spray application
PPFM populations established on both plants (Fig. 3),
except for the combination HSC5/wheat/no wetting agent,
where the bacteria were not found from the third week on
(Fig. 3B). In clover plants, the use of a wetting agent in
3.2. Rhizosphere and phyllosphere colonization after seed
bacterization
Both the gfp-tagged isolates tested readily colonized the
roots of the two plant species. Colonization of winter
wheat and red clover was comparable. The populations
of the tagged strains remained relatively stable during
the ¢rst 2 weeks, but counts decreased during the third
and fourth weeks (Fig. 2). Counts of both mutants in
the roots 8 weeks after sowing were never less than 103
CFU (g FW)31 . Visualizing the spatial distribution along
the root system by impressing the roots on AMS-Kan agar
plates, bacterial colonies were distributed along all the
length of the root system 5 days after seed germination
(result not shown).
No plant species speci¢city in colonization between the
isolate from winter wheat (Mb49) and the one from red
clover (HSC5) was apparent.
The seed inoculation experiment showed that PPFMs
are able to colonize plant root systems after seed bacterization as demonstrated both by the plate count (Fig. 2),
the root impression on AMS-Kan agar (results not shown)
and the confocal microscope images (see below, Fig.
6C,D). It is not yet clear to what extent PPFM bacteria
FEMSEC 1621 20-2-04
Fig. 3. Leaf colonization of red clover (A) and winter wheat (B) by
PPFM mutants after spray application. Bacterial counts were made in
bulked leaf samples collected 1 week after leaf spraying of bacterial cell
suspension in water with and without wetting agent (wa). Isolate Mb49
from winter wheat; isolate HSC5 from red clover. Error bars are V S.D.
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Z.S. Omer et al. / FEMS Microbiology Ecology 47 (2004) 319^326
newly emerged leaf D and leaf E was obtained after the
second and the third weeks, as shown in Fig. 5. Bacterial
colonies were either not detected or below the detection
limit.
The results show that the PPFMs applied persist in the
phyllosphere with a positive e¡ect of the wetting agent.
Besides the e¡ect of promoting the bacterial attachment
to the leaf surface, the wetting agent could provide a positive e¡ect by increasing the amount of methanol on the
leaf surface. In fact, Corpe and Rheem [3] reported an
increased amount of free methanol in white clover treated
with Triton X-100. The overall results of the inoculation
experiments give evidence of a limited capability of the
bacteria to occupy a new leaf surface. For clover, the
colonization of the next emerging leaf after spraying
(Fig. 4) might be the consequence of the fact that the
apical bud was actually already present at the time of
spraying. The role of humidity droplets or air-borne soil
granules carrying PPFMs as a way of intra- and inter-
Fig. 4. Colonization of speci¢c leaves in red clover followed for 2 weeks
after spray inoculation. A, cotyledons; B, unifoliate leaf; C, ¢rst trifoliate leaf; D, second trifoliate leaf. Isolate Mb49 from winter wheat; isolate HSC5 from red clover ; wa, wetting agent. Error bars are V S.D.
the bacterial suspension resulted in higher bacterial levels
during the course of the experiment (Fig. 3A). In the
wheat plants the overall amount of PPFMs was lower,
but the wetting agent also in this case resulted in increased
bacterial counts at all sampling times. The isolate HSC5
was also dependent on the wetting agent for surviving on
the wheat phyllosphere after the second week (Fig. 3B).
In order to estimate the colonization both on sprayed
leaves and on newly emerging unsprayed leaves, bacterial
counts were also made on single leaf samples based on leaf
age. In red clover, bacterial levels 1 week after spraying
showed that the two mutants were able to colonize the
¢rst true leaf not present at the time of spraying (leaf C
in Fig. 4) in addition to the cotyledons and the unifoliate
leaves (leaves A and B respectively in Fig. 4), which were
directly sprayed. Bacterial levels in the cotyledons (leaf A)
were signi¢cantly higher than in both leaves B and C in
the water-based spray treatment. However, when the wetting agent was used, there was no di¡erence in bacterial
counts between the cotyledons and leaf B. Two weeks
after the treatment, the bacterial levels decreased. In the
newly emerged leaf D, bacterial colonies were either not
found or below the detection limit.
Similarly, in wheat plants both mutants were able to
colonize the newly emerged leaves C in addition to the
coleoptiles and the ¢rst leaf originally sprayed (Fig. 5).
The wetting agent positively a¡ected the colonization of
leaf C. On the other hand, no further colonization of the
FEMSEC 1621 20-2-04
Fig. 5. Colonization of speci¢c leaves of winter wheat followed for
3 weeks after shoot spraying. Isolate Mb49 from winter wheat; isolate
HSC5 from red clover; wa, wetting agent. Error bars are V S.D. *Bacterial count below the detection limit.
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Z.S. Omer et al. / FEMS Microbiology Ecology 47 (2004) 319^326
325
Fig. 6. Confocal microscope images of red clover leaves (A,B) and roots of clover (C) and wheat (D) colonized by gfp-tagged PPFMs. A and B include
orthogonal sections of a stack of images. Arrows in A and B show the deep colonization into the leaf grooves. s, stomata. Scale bars: A, 40 Wm; B, 20
Wm; C, 8 Wm; D, 20 Wm.
plant spreading of PPFMs [20] is well in line with these
results.
The two plant species considered in this study showed a
signi¢cant di¡erence in the size and variability of the
respective PPFM populations. Clover phyllosphere was
more densely and more consistently colonized than wheat.
However, since the plant inoculation experiments carried
out showed no intrinsic preferences, or speci¢city, for
plant species in the isolates tested (Figs. 3^5), these di¡erences appear to be dependent mainly on the plant. Such
plant species e¡ects on bacterial population size and variability have already been described [16], and it has also
been reported that variability of bacterial population levels
isolated from upright, vertical plants, like wheat, is higher
than that from leaves that grow closer to the ground [16].
3.4. Site localization study of gfp-tagged mutants by
confocal microscopy
By use of CSLM, gfp-tagged PPFMs were visualized on
leaves and roots. On red clover, PPFMs colonize the leaf
surface by forming large aggregates and by occupying the
grooves between the epidermal cells (Fig. 6A,B). Although
FEMSEC 1621 20-2-04
the bacteria can penetrate deep into the epidermal grooves
of the leaves (see arrows in Fig. 6A,B), no colonization of
the lower tissue layers was apparent. On red clover the
bacteria were not aggregated and were found mainly on
the root hairs (Fig. 6C).
GFP expression in Methylobacterium extorquens was
earlier described by Figueira et al. [21] who reported transformation with a plasmid carrying gfp under the control of
the LacZ promoter and the methane monooxygenase promoter. In this study, the two selected isolates were tagged
in the chromosome by means of the mini-Tn5 transposon,
resulting in green £uorescing bacteria with a fairly low
intensity of £uorescence. This was probably due to the
quenching e¡ect of the bacterial pigments. As a consequence, the high background £uorescence of the wheat
material and the lack of bacterial aggregation prevented
easy visualization of tagged PPFMs on this plant. Nonetheless, it appears that on wheat leaves, the bacteria do
not form aggregates to the same extent as on clover leaves
(results not shown) and on wheat roots they are scattered
as on clover roots (Fig. 6D). On clover leaves, the tagged
PPFM bacterial strains did not appear to colonize the
stomatal regions as was expected. The stomata are the
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Z.S. Omer et al. / FEMS Microbiology Ecology 47 (2004) 319^326
site of methanol release from the plant [22] and hence were
expected to be the primary site of colonization for PPFMs
where these rely on plant methanol for growth. However,
this last assumption has not yet been proved.
The correlation of the presence of aggregates and a
higher PPFM population in clover than in wheat corroborates the hypothesis that the formation of large aggregates or bio¢lms on the leaf surfaces contributes to the
success of phyllosphere colonization [17]. Bio¢lms are normally formed by exopolysaccharides that incorporate the
bacteria and protect them from desiccation and other
physical stresses. The deep penetration of the bacterial
aggregates into the grooves between the epidermal cells
(illustrated in Fig. 6A,B) might represent the formation
of ‘protected sites’ (protected from surface sterilization
[23]) which might explain why PPFMs have sometimes
been considered endophytes [24].
[9]
[10]
[11]
[12]
[13]
[14]
Acknowledgements
[15]
This work was supported by Stiftelsen Lantbruksforskning (SLF) and by the Foundation for Strategic Environmental Research (MISTRA).
[16]
[17]
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Cyaan Magenta Geel Zwart