APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2009, p. 748–757
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 3
Genome Survey and Characterization of Endophytic Bacteria Exhibiting a
Beneficial Effect on Growth and Development of Poplar Trees?†
Safiyh Taghavi,1Craig Garafola,1Se ´bastien Monchy,1Lee Newman,2Adam Hoffman,2Nele Weyens,3
Tanja Barac,3Jaco Vangronsveld,3and Daniel van der Lelie1*
Brookhaven National Laboratory (BNL), Biology Department, Building 463, Upton, New York 11973-50001; University of South Carolina,
Arnold School of Public Health, 921 Assembly Street, Columbia, South Carolina 29208, and Savannah River Ecology Laboratory,
Aiken, South Carolina 298022; and Universiteit Hasselt, Department of Environmental Biology, CMK,
Universitaire Campus Building D, B-3590 Diepenbeek, Belgium3
Received 29 September 2008/Accepted 21 November 2008
The association of endophytic bacteria with their plant hosts has a beneficial effect for many different plant
species. Our goal is to identify endophytic bacteria that improve the biomass production and the carbon
sequestration potential of poplar trees (Populus spp.) when grown in marginal soil and to gain an insight in the
mechanisms underlying plant growth promotion. Members of the Gammaproteobacteria dominated a collection
of 78 bacterial endophytes isolated from poplar and willow trees. As representatives for the dominant genera
of endophytic gammaproteobacteria, we selected Enterobacter sp. strain 638, Stenotrophomonas maltophilia
R551-3, Pseudomonas putida W619, and Serratia proteamaculans 568 for genome sequencing and analysis of their
plant growth-promoting effects, including root development. Derivatives of these endophytes, labeled with gfp,
were also used to study the colonization of their poplar hosts. In greenhouse studies, poplar cuttings (Populus
deltoides ? Populus nigra DN-34) inoculated with Enterobacter sp. strain 638 repeatedly showed the highest
increase in biomass production compared to cuttings of noninoculated control plants. Sequence data combined
with the analysis of their metabolic properties resulted in the identification of many putative mechanisms,
including carbon source utilization, that help these endophytes to thrive within a plant environment and to
potentially affect the growth and development of their plant hosts. Understanding the interactions between
endophytic bacteria and their host plants should ultimately result in the design of strategies for improved
poplar biomass production on marginal soils as a feedstock for biofuels.
Endophytic bacteria are bacteria that reside within the living
tissue of their host plants without substantively harming it (19,
26). They are ubiquitous in most plant species, latently residing
or actively colonizing the tissues. The diversity of cultivable
bacterial endophytes is exhibited not only in the variety of
plant species colonized but also in the many taxa involved, with
most being members of common soil bacterial genera such as
Enterobacter, Pseudomonas, Burkholderia, Bacillus, and Azos-
pirillum (21, 23). Endophytic bacteria have several mechanisms
by which they can promote plant growth and health. These
mechanisms are of prime importance for the use of plants as
feedstocks for biofuels and for carbon sequestration through
biomass production. This is vital when considering the aim of
improving biomass production of marginal soils, thus avoiding
competition for agricultural resources, which is one of the
critical socioeconomic issues of the increased use of biofuels.
Like rhizosphere bacteria, endophytic bacteria have been
shown to have plant growth-promoting activity that can be due
to the production of phytohormones, enzymes involved in
growth regulator metabolism, such as ethylene, 1-aminocyclo-
propane-1-carboxylic acid (ACC) deaminase, auxins, indole-3-
acetic acid (IAA), acetoin, 2,3-butanediol, cytokinins (3, 13–15,
20, 30), or combinations thereof. They can also improve plant
growth via the fixation of nitrogen (diazotrophy) (9, 38).
Typical examples of marginal soils include soils that have
deteriorated due to the presence of heavy metals or organic
contaminants. These are often soils with a history of industrial,
military, or mining activities. Endophytic bacteria can assist
their host plants in overcoming phytotoxic effects caused by
environmental contamination (5, 11, 12, 36), which is of direct
relevance for waste management and pollution control via
phytoremediation technologies. When nonsterile poplar cut-
tings (Populus trichocarpa ? deltoides cv. Hoogvorst) were in-
oculated with the endophyte Burkholderia cepacia VM1468, a
derivative of B. cepacia Bu72 which possesses the pTOM-Bu61
plasmid coding for a constitutively expressed toluene degrada-
tion pathway, it was observed that in addition to decreasing the
phytotoxicity and releasing toluene, strain VM1468 also con-
siderably improved the growth of poplar trees in the absence of
toluene (36). This observation, which was the first of its kind
for poplar trees, prompted us to further study the poplar tree-
associated beneficial endophytic bacteria in order to improve
the overall performance of poplar trees, as it can enhance
multiple applications, including biomass production, carbon
sequestration, and phytoremediation. This was done by screen-
ing endophytic bacteria for their plant growth-promoting ca-
pabilities toward poplar trees by performing colonization stud-
ies with gfp-labeled strains, by examining their metabolic
properties, and by initiating the genome sequencing of several
* Corresponding author. Mailing address: BNL, Biology Depart-
ment, Building 463, Upton, NY 11973-5000. Phone: (631) 344-5349.
Fax: (631) 344-3407. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://aem
?Published ahead of print on 5 December 2008.
MATERIALS AND METHODS
Isolation of endophytic bacteria. Root and shoot samples were collected from
the 10-year-old hybrid poplar tree H11-11 (Populus trichocarpa ? P. deltoides)
that had been growing in the presence of carbon tetrachloride (12 ppm homo-
geneously) for 8 years at an experimental site in Washington State. In addition,
native willow (Salix gooddingii) material was collected from 5-year-old native
plants that had been growing in the presence of both trichloroethylene (18 ppm)
and carbon tetrachloride (12 ppm) for 5 years. Cuttings were removed from the
plants with clippers that were washed with ethanol between cuts and placed in
acetone-rinsed volatile organic analysis vials which were placed on ice for ship-
ment from the field. Roots and shoots were treated separately. Fresh root and
shoot samples were vigorously washed in distilled water for 5 min, surface
sterilized for 5 min in a solution containing 1% (wt/vol) active chloride (added as
a sodium hypochlorite [NaOCl] solution) supplemented with 1 droplet Tween 80
per 100 ml solution, and rinsed three times in sterile distilled water. A 100-?l
sample of the water from the third rinse was plated on 869 medium (25) to verify
the efficiency of sterilization. After sterilization, the roots and shoots were mac-
erated in 10 ml 10 mM MgSO4using a Polytron PT1200 mixer (Kinematica A6).
Serial dilutions were made, and 100-?l samples were plated on nonselective
media in order to test for the presence of the endophytes and their character-
16S rRNA gene amplification, amplified 16S rRNA gene restriction analysis
(ARDRA), sequencing, and strain identification. Total genomic DNA of endo-
phytic bacteria was isolated as described previously (7). 16S rRNA genes were
PCR amplified using the standard 26F-1392R primer set (2).
For ARDRA, aliquots of the PCR products were digested overnight at 37°C
with HpyCH4IV in 1? NEB buffer 1 (New England Biolabs, Beverly, MA).
ARDRA patterns were grouped, and clones with representative patterns were
selected for sequencing.
Purified PCR products (QIAquick columns) of 16S rRNA genes were se-
quenced using the Prism BigDye terminator sequencing kit (Applied Biosystems,
Foster City, CA) with 100 ng of template DNA. The extended sequences were
obtained with universal primers 26F and 1392R. Taxonomic classifications were
determined according to Wang et al. (44) at the Ribosome Database Project II
(http://rdp.cme.msu.edu/index.jsp). The sequences used for identification of the
cultivable endophytes are available in the GenBank database (www.ncbi.nlm.nih
.gov) under accession numbers EU340901 through EU340978.
Screening for metabolic properties. Bacteria were screened for their carbon
utilization in Schatz minimal salt medium (31) supplemented with different
carbon sources, which were added at 2 g/liter. As positive controls, strains were
grown in Schatz minimal salt medium supplemented with C-mix (per liter of
medium: 1.3 ml glucose 40%, 0.7 ml lactate 50%, 2.2 ml gluconate 30%, 2.7 ml
fructose 20%, and 3 ml 1 M succinate). To determine IAA production, strains
were grown in Schatz medium plus C-mix supplemented with 100 ?g/ml L-
tryptophan. IAA concentrations were determined by the Salkowsky reaction
(24). In order to test for growth on toluene as the sole carbon source, bacteria
were plated on Schatz medium and incubated for 7 days at 30°C in sealed 10-liter
vessels with the addition of 600 ?l toluene. To test for autotrophy and nitrogen
fixation, bacteria were inoculated in Schatz medium without a carbon or nitrogen
source (omit NH4NO3), respectively. ACC deaminase activity was tested, with
ACC as the sole nitrogen source (43). For reproducibility, all experiments were
done in triplicate, starting from isolated colonies.
Inoculation of poplar trees with endophytic bacteria and analysis of plant
growth. Inocula (250-ml culture) were prepared by growing endophytic bacteria
in 1/10-strength 869 medium (25) at 30°C on a rotary shaker until a cell concen-
tration of 109CFU/ml was reached (optical density at 660 nm [OD660] of 1). The
cells were collected by centrifugation, washed twice in 10 mM MgSO4, and
suspended in 1/10 of the original volume (in 10 mM MgSO4) to obtain an
inoculum with a cell concentration of 1010CFU/ml. Per microbial strain tested,
seven cuttings from poplar (Populus deltoides ? P. nigra) DN-34 of approxi-
mately 30 cm were weighed and placed in a 1-liter beaker containing 0.5 liter of
a half-strength sterile Hoagland’s nutrient solution (5), which was refreshed
every 3 days. The cuttings were allowed to root for approximately 4 weeks until
root formation started. Subsequently, a bacterial inoculum was added to each jar
at a final concentration of 108CFU/ml in half-strength Hoagland’s solution.
After 3 days of incubation, cuttings were weighed and planted in nonsterile sandy
soil and placed in the greenhouse with a constant temperature of 22°C and 14 h
light-10 h dark cycle with photosynthetic active radiation of 165 mmol/m2s. After
10 weeks, plants were harvested, and their total biomass, their increase in bio-
mass, and the biomass of different plant tissues were determined. Data were also
collected from noninoculated control plants. Growth indexes were calculated as
(Mt ? M0)/M0 after 10 weeks of growth in the presence or absence of endo-
phytic inoculum, where M0 is the plant’s weight (g) at week 0 and Mt is the
plant’s weight (g) after 10 weeks. The statistical significance of the results was
confirmed at the 5% level using the Dunnett test.
To determine the effects of endophytic bacteria on the rooting of poplar
DN-34, cuttings were treated as described above, except that the endophytic
inoculum was added from day 1.
Construction and imaging of green fluorescent protein (GFP)-labeled
endophytes. In order to follow endophytic colonization of poplar trees, we
successfully labeled Enterobacter sp. strain 638, Pseudomonas putida W619 (36),
and Serratia proteamaculans 568 with gfp using Escherichia coli S17-1/?pir
(pUT::miniTn5-Km-gfp) (37) as a donor in conjugation (35). Kanamycin (100
?g/ml)-resistant transconjugants were selected on 284 minimal medium (32)
complemented with a carbon mix (a mixture of glucose, gluconate, lactate,
succinate, and acetate was added at 0.05% [wt/vol] each) and subsequently tested
for gfp expression under UV light.
The stability of the gfp insertions was verified by growing individual transcon-
jugants for 100 generations on nonselective 869 medium (25). One hundred
individual colonies were grown on nonselective medium and subsequently replica
plated on 869 minimal medium supplemented with kanamycin, after which they
were tested for gfp expression. All gfp-labeled strains gave transconjugants that
stably maintained the insertion (less than 1% loss of the gfp marker after growth
for 100 generations under nonselective conditions). No gfp-expressing derivatives
of Stenotrophomonas maltophilia R551-3 and Methylobacterium populi BJ001
were obtained. S. maltophilia R551-3 was found to possess natural resistance to
kanamycin and tetracycline, the two antibiotics available to select gfp-containing
minitransposons. For M. populi BJ001, neither transformation nor horizontal
gene transfer could be successfully used to introduce the gfp-containing mini-
To determine the sites of miniTn5-Km2-gfp insertion, genomic DNA was
isolated (7) and digested with HpyCH4IV, which has no recognition site between
the 5? end of gfp and the upstream end of the miniTn5 transposon part. After
digestion, HpyCH4IV was heat inactivated for 20 min at 65°C. Subsequently, the
restriction fragments were ligated to a Y-shaped linker cassette, which was
obtained by annealing two oligonucleotides with sequences 5?-TTTGGATTTG
CTGGTCGAATTCAACTAGGCTTAATCCGACA-3? and 5?-CGTGTCGGA
TTAAGCCTAGTTGAATTTATTCCTATCCCTAT-3? as described previously
(42). The ligation mixture was purified using the GFX PCR DNA and gel band
purification kit (GE Healthcare Biosciences) and used as a template for linear
amplification, using a single complementary primer pointing outward from the 5?
end of gfp (GFP primer, 5?-GAAAAGTTCTTCTCCTTTAC-3?). The linear
amplification results in the repair of the Y-shaped linker cassette only for those
fragments that contain the gfp insertion region. Subsequently, PCR was per-
formed using the GFP primer plus the linker primer (5?-GGATTTGCTGGTC
GAATTCAAC-3?), which will only hybridize to the repaired linker. For each of
the transconjugants tested, this resulted in the amplification of a single DNA
fragment, indicative of a single insertion of miniTn5-Km2-gfp. Sequence analysis
allowed for determining the location of the miniTn5-Km2-gfp insertions as fol-
lows: position 341748 of the P. putida W619::gfp7 chromosome within ORF326,
which encodes a putative heavy metal-translocating P-type ATPase (CadA like);
position 8455 of plasmid pENT628-1 of Enterobacter sp. strain 638::gfp7 in the
noncoding region of a two-component regulatory system; and position 27524 of
the 46,804-bp plasmid pSER568-1 of S. proteamaculans 568::gfp1 within
ORF4938, which encodes a putative HNH endonuclease. None of the insertions
seemed to have occurred in functions with obvious roles in plant-microbe inter-
The Axiovert 200 M (Zeiss) fluorescence microscope, equipped with an Axio-
Cam MRm charge-coupled-device camera and ApoTome, was used to visualize
gfp-labeled bacteria on the surface of and inside the plant tissue. Images were
acquired with Zeiss AxioVision software. The colors on the image are pseudo-
colors. The green channel was acquired with Zeiss filter set 10 (excitation, 450 to
490 nm; beam splitter, 510 nm; emission, 515 to 565 nm), and the red channel was
acquired with Zeiss filter set 00 (excitation, 530 to 585 nm; beam splitter, 600 nm;
emission, ?615 nm).
After being harvested, plant roots were rinsed with 10 mM MgSO4. Visual-
ization of gfp expression proved to be difficult due to autofluorescence from the
plant tissue itself and was achieved by counterstaining the tissue section with
0.05% methyl violet for 30 s, which caused the plant cells to fluoresce red under
near-UV light (11).
Genome sequencing. Genome sequencing was carried out at the Joint Genome
Institute (DOE, Walnut Creek, CA) for Enterobacter sp. strain 638 (http:
//genome.jgi-psf.org/finished_microbes/ent_6/ent_6.home.html), P. putida W619
(http://genome.jgi-psf.org/finished_microbes/psepw/psepw.home.html), S. pro-
teamaculans 568 (http://genome.jgi-psf.org/finished_microbes/serpr/serpr.home
VOL. 75, 2009PLANT GROWTH-PROMOTING ENDOPHYTES FROM POPLAR TREES749
.html), and S. maltophilia R551-3 (http://genome.jgi-psf.org/finished_microbes
/stema/stema.home.html). Metabolic functions were identified using the
integrated microbial genomes (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi) sys-
tem (22) and with the basic local alignment search tool (BLAST) (1). Metabolic
pathways were constructed using PRIAM predictions mapped on the KEGG
Isolation and characterization of endophytic bacteria from
poplar and willow trees. Endophytic bacteria were isolated un-
der aerobic conditions from surface-sterilized root and stem sam-
ples taken from hybrid poplar tree H11-11 and native willow
(Salix gooddingii) that were grown in a silty loam soil with ground-
morphological characteristics, 78 strains were selected for identi-
fication. Their total genomic DNA was extracted and used to
amplify the 16S rRNA gene. ARDRA with HpyCH4IV was used
to determine closely related clones, after which representative
16S rRNA genes were sequenced for species identification. A
detailed breakdown of the cultivable endophytic community com-
position is presented in Fig. 1. The majority of the isolated strains
(71%) belonged to Gammaproteobacteria, with Serratia spp., Ser-
ratia plymuthica, Serratia proteamaculans, and Rahnella spp. being
the most frequently found. Other dominant gammaproteobacte-
ria included Pseudomonas spp. and Enterobacter spp. The Acti-
nobacteria (15% of the population) were dominated by Rhodo-
coccus spp. The presence of Stenotrophomonas maltophilia is also
noteworthy, as this species represents an increasing medical issue
of multidrug resistance (6) and seems to be well adapted to live in
association with both human and plant hosts.
Screening of endophytic bacteria for plant growth-promot-
ing properties. Selected cultivable endophytic gammaproteobac-
teria found in poplar trees, isolated as part of this study or pre-
viously described and which represent the major phylogenetic
groups as identified in Fig. 1, were tested for their capacity to
improve the growth of their host plants. The selected strains were
the poplar endophytes Enterobacter sp. strain 638 (this study), S.
maltophilia R551-3 (36), P. putida W619 (36), S. proteamaculans
Serratia proteamaculans 17
Serratia plymuthica 12
Serratia sp. 1.2
Rahnella sp. 17
Enterobacter amnigenus 6.1
Enterobacter sp. 2.4
Pseudomonas sp. 6.1
Pseudomonas rhizosphaerae 1.2
Kluyvera cochleae 2.4
Stenotrophomonas maltophilia 2.4
Buttiauxella gaviniae 1.2
Erwinia sp. 1.2
Rhodococcus sp. 6.1
Rhodococcus equi. 6.1
Leifsonia xyli. 1.2
Agromyces sp. 1.2
Agrobacterium rhizogenes 4.9
Agrobacterium tumefaciens 1.2
Rhizobium sp. 1.2
Burkholderia sp. 3.7
Alcaligenes sp. 1.2
Staphylococcus sp. 1.2
Bacillus barbaricus 1.2
FIG. 1. Taxonomic breakdown of 16S rRNA gene sequences of the cultivable endophytic community composition as isolated from hybrid
poplar H11-11 and willow trees. Taxonomic classifications were determined according to Wang et al. (44). The central pie shows percentages by
phyla; each outer annulus progressively breaks these down to finer taxonomic levels, with families, genera, and species in the outermost annuli.
Numbers indicate the relative abundance, expressed as a percentage, of the different taxonomic groups.
750TAGHAVI ET AL.APPL. ENVIRON. MICROBIOL.
568 (this study), and M. populi BJ001 (40, 41). Burkholderia ce-
pacia Bu72, an endophyte originally isolated from yellow lupine
which was found to have plant growth-promoting effects on pop-
lar trees (36), and Cupriavidus metallidurans CH34 (also referred
to as Ralstonia metallidurans CH34) (27), a typical soil bacterium
with no known plant growth-promoting effects, were included as
positive and neutral controls, respectively. Also, noninoculated
cuttings were used as controls.
After root formation in hydroponic conditions and subse-
quent endophytic inoculation, the poplar DN-34 cuttings were
planted in a marginal sandy soil and allowed to grow for 10
weeks, after which the plants were harvested and their bio-
masses were determined. After 10 weeks of growth, poplar
trees inoculated with M. populi BJ001 had less new biomass
than the controls (Fig. 2) (P ? 0.05). Poplar cuttings inocu-
lated with Enterobacter sp. strain 638 (P ? 0.018) and B. cepa-
cia BU72 (P ? 0.042) showed statistically better growth than
the control plants (Fig. 2), as was reflected by their growth
indexes. The plant growth-promoting effects of Enterobacter sp.
strain 638 and B. cepacia BU72 were reproducible in indepen-
dently performed experiments.
Under the greenhouse conditions tested, no differences in
growth indexes were found between those of the noninocu-
lated control plants and those for plants inoculated with S.
maltophilia R551-3, P. putida W619, and S. proteamaculans
568; their growth was comparable to that observed for plants
inoculated with C. metallidurans CH34. Also, control plants
and plants inoculated with the endophytic bacteria appeared
healthy, except for plants inoculated with M. populi BJ001,
which showed signs of stress, including chlorosis of the leaves.
Effects of endophytic bacteria on poplar root development.
During hydroponic growth of poplar DN-34 cuttings and be-
fore endophytic inoculation occurred, difficulties with root for-
mation were observed. After endophytic inoculation and sub-
sequent growth in soil, we noticed that the root systems of
inoculated poplar cuttings were often denser with many fine
roots compared to those of the noninoculated control plants.
To further test the effects of endophytic bacteria on root de-
velopment, rooting experiments were performed in the pres-
ence and absence of gfp-labeled derivatives of S. proteamacu-
lans 568, P. putida W619, and Enterobacter sp. strain 638. Root
formation was very slow for noninoculated plants. In contrast,
for cuttings that were allowed to root in the presence of the
selected endophytes, root formation was initiated within 1
week, and shoot formation was more pronounced compared to
that of the noninoculated plants (Fig. 3A). After 10 weeks, root
formation for the noninoculated controls was still poor; how-
ever, for plants inoculated with S. proteamaculans 568, roots
and shoots were well developed (Fig. 3B). Similar effects on
root and shoot development were repeatedly noticed for plants
inoculated with P. putida W619 and Enterobacter sp. strain 638
(see Fig. S-1A and B in the supplemental material).
FIG. 2. Growth indexes for poplar cuttings inoculated with differ-
ent endophytic bacteria. Growth indexes were determined 10 weeks
after the inoculating and planting of the cuttings in sandy soil. Per
condition, seven plants were used. Plants were grown in the green-
house. Noninoculated plants were used as references. Bars indicate
standard errors. Growth indexes were calculated as (Mt ? M0)/M0
after 10 weeks of growth in the presence or absence of endophytic
inoculum. M0, plant’s weight (g) at week 0; Mt, plant’s weight (g) after
10 weeks. The statistical significance of the increased biomass produc-
tion of inoculated plants, compared to that of noninoculated control
plants, was confirmed at the 5% level (**) using the Dunnett test.
FIG. 3. Effects of S. proteamaculans 568 on the rooting and shoot formation of poplar DN-34. Plants were incubated hydroponically in
half-strength Hoagland’s solution in the absence (control) or presence (568) of strain 568. Root and shoot development are presented after 1 week
(A) and 10 weeks (B).
VOL. 75, 2009 PLANT GROWTH-PROMOTING ENDOPHYTES FROM POPLAR TREES 751
Fluorescence microscopy was used to visualize the inter-
nal colonization of the plant roots by the gfp-labeled strains,
confirming their endophytic behavior (Fig. 4). For P. putida
W619, the formation of microcolonies on the root surface
was observed in addition to internal colonization (Fig. 4A
and B). Interestingly, these colonies were absent on the root
surfaces of plants inoculated with S. proteamaculans 568 and
Enterobacter sp. strain 638, where only internal colonization
was observed (Fig. 4C and D). No gfp expression was de-
tected for roots from noninoculated control plants (results
Bacterial properties hypothesized to be important for colo-
nization and plant growth promotion. In order to better
understand their plant growth-promoting properties, we
screened endophytic bacteria for properties related to phy-
tohormone production and the metabolism of plant growth-
regulating compounds, as well as the utilization of different
carbon sources, including plant biomass-derived sugars and
plant metabolites. These compounds included IAA produc-
tion from tryptophan and the metabolism of phenylacetic
acid, 4-aminobutyrate (GABA), and ACC, which plants pro-
duce as a precursor of stress ethylene. Burkholderia vietna-
miensis Bu61, a derivative of the environmental strain B.
vietnamiensis G4 that constitutively expresses toluene and
trichloroethylene degradation (33, 34), was included for
comparison with B. cepacia Bu72. The results are presented
in Table 1.
None of the endophytes tested was able to grow autotrophi-
cally or able to fix nitrogen. The Burkholderia strains were able
to grow in ACC as the sole nitrogen source. These strains, as
well as S. proteamaculans 568 and P. putida W619, were also
able to metabolize phenylacetic acid and GABA. GABA could
also support the growth of M. populi BJ001. All strains pro-
duced IAA, as was determined using the method of Salkowski
(17), with the highest levels observed for P. putida W619 and
M. populi BJ001. Some interesting differences were observed
when comparing the Burkholderia strains: in contrast to the
environmental strain B. vietnamiensis Bu61, B. cepacia Bu72
was able to utilize arbutin, salicin, pectin, trehalose, and cel-
lobiose, compounds typically found in poplar and willow trees.
Also, the other endophytic strains tested seem well adapted to
utilize a broad spectrum of plant-derived compounds as carbon
sources. Only Enterobacter sp. strain 638 and S. proteamaculans
568 were able to grow on lactose as the sole carbon source,
which is consistent with the presence of a lacZ gene on their
FIG. 4. Endophytic colonization of the poplar DN-34 roots by gfp-labeled derivatives of S. proteamaculans 568, Enterobacter sp. strain 638, and
P. putida W619. (A) Colonization of the surface of a poplar root by a gfp-labeled derivative of P. putida W619. The picture was taken by
fluorescence microscopy. Arrows indicate the positions of microcolonies on the root surface. (B) Interior view of a translateral section of a poplar
root colonized by a gfp-labeled derivative of P. putida W619. The picture was taken with the help of the apotome feature of the fluorescence
microscope. Arrows indicate the zones of dense interior colonization. (C and D) Interior views of a section of a poplar root colonized by a
gfp-labeled derivative of S. proteamaculans 568 (C) and Enterobacter sp. strain 638 (D). The root tissue was stained with 0.05% methyl violet to
752TAGHAVI ET AL.APPL. ENVIRON. MICROBIOL.
Preliminary analysis of the genome sequences of endophytic
bacteria for plant growth-promoting functions. Genome se-
quencing of Enterobacter sp. strain 638, S. maltophilia R551-3,
P. putida W619, and S. proteamaculans 568 was exploited for
properties related to phytohormone production and the me-
tabolism of plant sugars and growth-regulating compounds. A
schematic overview of the pathways required for the synthesis
of the plant growth-promoting compounds and their distribu-
tion among these endophytes is presented in Fig. 5.
Acetoin and 2,3-butanediol synthesis. It was shown that a
blend of volatile compounds, especially 3-hydroxy-2-butanone
(acetoin) and 2,3-butanediol, emitted by rhizobacteria can en-
hance plant growth (29, 30). Acetolactate synthase (AlsS) and
acetolactate decarboxylase (AlsD) catalyze the two-step con-
version from pyruvate to acetoin, which can be converted into
2,3-butanediol, either by the bacteria or by the host plant. The
alsDS acetoin synthesis pathway was present in S. proteamacu-
lans 568 and Enterobacter sp. strain 638, which also can convert
acetoin into diacetyl, a compound whose role in plant growth
promotion is unknown. The acetolactate decarboxylase gene
alsD was lacking in S. maltophilia R551-3 and P. putida W619.
IAA synthesis. Endophytic bacteria also enhance plant
growth through the synthesis of the plant auxin IAA, whose
production was the most pronounced for P. putida W619. IAA
synthesis from tryptophan can occur via three alternative path-
ways (Fig. 5), two of which were present in strain W619. The
first pathway involves a tryptophan-2-monooxygenase (IaaM)
that oxidizes tryptophan to indole-3-acetamide and an in-
doleacetamide hydrolase (IaaH) that produces IAA. Genome
analysis revealed that although a putative iaaH gene was
present in all strains except Enterobacter sp. strain 638, the
putative tryptophan 2-monooxygenase could only be found in
P. putida W619 and B. vietnamiensis Bu61. P. putida W619
further has the complete pathway to synthesize IAA via
tryptamine and indole-3-acetaldehyde and lacks the trypto-
phan 2,3-dioxygenase (KynA), thus driving the conversion of
excess tryptophan to IAA synthesis instead of tryptophan me-
tabolism. Consistent with this high level of IAA production is
the presence of four genes encoding putative auxin carriers on
the W619 genome.
ACC metabolism. Putative ACC deaminase genes were
found in P. putida W619, Enterobacter sp. strain 638, and S.
proteamaculans 568. However, none of them coded for a pro-
tein that possessed the conserved amino acid signature char-
acteristic for a genuine ACC deaminase (16) (see Fig. S-2 in
the supplemental material). The lack of ACC deaminase ac-
tivity was confirmed by the inability of these strains to grow on
ACC as their sole nitrogen source (Table 1). Consistent with its
growth on ACC, B. vietnamiensis Bu61 coded for a putative
ACC deaminase with the correct signature.
?-Aminobutyrate and phenylacetic acid metabolism. S. pro-
teamaculans 568 and P. putida W619 metabolized GABA and
contained the complete pathway for GABA uptake and degrada-
TABLE 1. Screening of endophytic bacteria and related strains for their metabolic properties and functions that affect plant hormone
balances and growtha
Metabolic property or
568638 Bu61 Bu72 W619R551-3 BJ001
aThe endophytic bacteria S. proteamaculans 568, Enterobacter strain 638, B. cepacia Bu72, P. putida W619, S. maltophilia R551-3, and M. populi BJ001 and the soil
bacterium B. vietnamiensis Bu61 were tested for their metabolic properties. Cultures were inoculated at OD660of 0.01. Growth of the cultures was determined by an
increase of OD660, which was followed until the cultures had reached stationary growth phase or after 5 days. ???, OD660of ?0.8; ??, OD660of 0.5 to 0.8; ?, OD660
of 0.25 to 0.5; ?/?, OD660of 0.1 to 0.25; ?, OD660of ?0.1. Experiments were carried out as independent triplicates.
bAutotrophy or nitrogen fixation was tested by growing the strains on minimal growth medium that lacked a carbon or nitrogen source, respectively. ACC deaminase
activity (ACC) was determined by testing the growth on 1-aminocyclopropane-1-carboxylic acid as either a carbon or nitrogen source. GABA and PAA metabolism
were determined by testing bacterial growth on these compounds as either a carbon or nitrogen source. The IAA concentrations produced by strains 568, 638, Bu61,
Bu72, W619, R551-3, and BJ001 were 3.23, 3.90, 2.57, 2.38, 29.39, 2.98, and 9.38 ?g/ml, respectively. These concentrations were determined using the method of
Salkowski (17) and calculated for a culture OD660of 1.0.
cBrown color formation in the medium.
dOrange color formation in the medium.
eYellow color formation in the medium.
VOL. 75, 2009PLANT GROWTH-PROMOTING ENDOPHYTES FROM POPLAR TREES 753
R551-3 lacked the ?-aminobutyrate transaminase (EC 22.214.171.124),
while the ?-aminobutyrate permease (COG1113) was absent in
Enterobacter sp. strain 638. S. proteamaculans 568, P. putida
W619, and B. vietnamiensis Bu61 were also able to grow on phe-
nylacetic acid, which is consistent with the presence of paa oper-
ons on their genomes.
PTS sugar uptake systems. Genes coding for phosphotrans-
ferase (PTS) sugar uptake systems were dominantly present in
Enterobacter strain 638 and S. proteamaculans 568, and their
phylogenetically assigned substrate specificity seems to be con-
sistent with their sugar metabolism (Table 1). Enterobacter
strain 638 and S. proteamaculans 568 possessed PTS belonging
to the ?-glucoside family (for the uptake of glucose, N-acetyl-
glucosamine, maltose, glucosamine, and ?-glucosides; seven
and four copies for strains 638 and 568, respectively), ?-glu-
coside family (for the uptake of sucrose, trehalose, N-acetyl-
muramic acid, and ?-glucosides; seven and five copies, respec-
tively), fructose family (for the uptake of fructose, mannitol,
mannose, and 2-O-?-mannosyl-D-glycerate; two copies in both
strains), and lactose family (for the uptake of lactose, cellobi-
ose, and aromatic ?-glucosides; six and three copies, respec-
tively). A copy of the glucitol family (for the uptake of glucitol
and 2-methyl-D-erythritol) was only found in S. proteamaculans
568. Both P. putida W619 and S. maltophilia R551-3 possessed
a single gene coding for a PTS from the fructose family.
The cultivable endophytic bacteria from poplar and willow
trees share many closely related strains, the majority of which
FIG. 5. Metabolic pathways involved in the production of plant growth hormones (IAA, diacetyl, acetoin, and 2,3-butanediol) found on the
genomes of selected endophytic bacteria. The metabolic pathways were constructed using PRIAM predictions mapped on the KEGG database
(http://www.genome.ad.jp). For each organism, differently colored arrows are used to indicate the presence of the putative pathways: S. maltophilia
R551-3 (red), P. putida W619 (green), B. vietnamiensis G4 (orange), Enterobacter sp. strain 638 (dark blue), and S. proteamaculans 568 (light blue).
Black arrows indicate known pathway steps that could not be identified. Dashed arrows correspond to the presence of putative enzymes (PRIAM
E value below 10?30). In the tryptophan-dependent IAA synthesis, the enzymes involved are tryptophan transaminase Lao1 (1), indolepyruvate
decarboxylase IpdC (2), indole-3-acetaldehyde dehydrogenase DhaS (3), tryptophan decarboxylase Dcd1 (4), amine oxidase (5), tryptophan
2-monooxygenase IaaM (6), deaminase IaaH (7), nitrile hydratase (8), nitrilase YhcX (9), and indole-3-acetaldehyde reductase AdhC (10). Besides
the production of IAA, the main pathway for tryptophan metabolism is via tryptophan 2,3-dioxygenase KynA (11). In butanoate metabolism, the
production of acetoin from pyruvate is catalyzed by the acetolactate synthase AlsS (12) and the acetolactate decarboxylase AlsD (13). The genome
of Enterobacter sp. strain 638 encodes the acetoin dehydrogenase ButA (14) that is able to catalyze the conversion of acetoin into diacetyl. It is
unknown if this compound has plant growth stimulatory effects. The 2,3-butanediol dehydrogenase ButB (15) involved in the conversion of acetoin
into 2,3-butanediol was not found encoded on the genomes of the endophytic bacteria but is present on the poplar genome.
754 TAGHAVI ET AL.APPL. ENVIRON. MICROBIOL.
belong to the Gammaproteobacteria (Fig. 1). The dominance of
gammaproteobacteria is consistent with previous observations
of the endophytic community diversity in poplar trees growing
on a benzene-, toluene-, ethylbenzene-, and xylene-contami-
nated site (28). However, in contrast to previous studies, we
also observed a significant number of Rhodococcus spp. (12%
of the cultivable strains), including Rhodococcus equi. As no-
ticed previously (36), we found that the highest number of
endophytic bacteria reside in the roots, with their numbers
declining in the stems, shoots, and leaves (results not shown).
Endophytic bacteria from poplar trees, representative of the
dominantly observed genera Enterobacter, Serratia, Stenotroph-
omonas, and Pseudomonas, were tested for their capacities to
improve growth of their poplar host. In addition, they were
screened for the production or metabolism of plant growth-
promoting compounds, phytohormones, and sugars. A better
understanding of their plant growth-promoting capabilities was
further obtained by initiating the sequencing of their genomes.
Enterobacter sp. strain 638 had the most-pronounced bene-
ficial effect on the development and growth of poplar cuttings.
This result was not only repeatable in our hands with P. del-
toides ? P. nigra DN-34 but also with the hybrid poplar clone
0P367 (Populus deltoides ? P. nigra) (significance level, P ?
0.05) (L. Newman, unpublished results). On the other hand,
while no significant plant growth-promoting effect was ob-
served for P. putida W619 with P. deltoides ? P. nigra DN-34,
strain W619 significantly (significance level, P ? 0.01) pro-
moted the growth of another hybrid poplar tree [Populus del-
toides ? (P. trichocarpa ? P. deltoides) cv. Grimminge] (N.
Weyens, J. Boulet, D. Adriaensen, J.-P. Timmermans, E. Prin-
sen, S. Van Oevelen, J. D’Haen, K. Smeets, D. van der Lelie,
S. Taghavi, and J. Vangronsveld, submitted for publication).
Also, the promiscuous plant growth-promoting effect of B.
cepacia Bu72 on poplar trees (this study and reference 36) and
yellow lupine (5) is noticeable. Therefore, before the applica-
tion of this concept to other poplar cultivars, preliminary stud-
ies to confirm the plant growth-promoting effect of the selected
endophyte are required.
The plant growth-promoting effect of Enterobacter sp. strain
638 might be explained by the presence of the putative alsDS
pathway for acetoin synthesis (Fig. 5), a potent plant growth-
promoting compound (29, 30). As for the rhizosphere bacte-
rium Bacillus amyloliquefaciens FZB42 (8), it was unclear
which function catalyzes the conversion of acetoin into 2,3-
butanediol. We assumed that acetoin (produced and released
by Enterobacter sp. strain 638 and S. proteamaculans 568) can
enter the poplar cells, where it can be converted into 2,3-
butanediol. Enterobacter sp. strain 638 also possesses a putative
acetoin reductase for synthesis of diacetyl, whose role in plant
growth promotion is unknown.
None of the other traits linked to plant growth regulation
were identified in Enterobacter sp. strain 638: the strain pro-
duces low levels of IAA, is unable to fix nitrogen, and lacks the
pathways to metabolize ACC, GABA, and phosphonoacetic
acid (PAA). Sequence analysis, however, revealed that Entero-
bacter sp. strain 638 contains a 157.7-kb plasmid, pENT638-1,
which is related to F plasmids found in other Enterobacteria-
ceae. Plasmids of this family are involved in host interaction
and virulence, such as the pFra plasmid of the plague microbe
Yersinia pestis (18). In pENT638-1, the pFra pathogenicity is-
land has been replaced by a 23-kb putative genomic island
(flanked by an integrase gene and having a GC content that is
significantly different than that of the rest of the plasmid). This
island contains a group of open reading frames with strong
homology to hypothetical proteins of Azotobacter vinelandii
AvOP, as well as to a putative srfABC operon, which is also
present in a horizontally acquired region of Salmonella spp.
and which is believed to be involved in virulence (46). Adjacent
to this region, a putative ndvB (8,532-bp) gene was located.
ndvB, which codes for a protein involved in the production of
?-(132)-glucan, is required by Sinorhizobium meliloti for bac-
terial invasion of the nodule (10). Many other genes involved
in plant invasion were present on pENT638-1—genes coding
for proteins with an autotransporter domain (type V secretion)
or virulence domains (agglutinin, pertactin, or adhesin). In
addition, plasmid pENT628-1 carries many relBE toxin/anti-
toxin systems, often located in the proximity of regions that
presumably play a role in the successful interaction between
Enterobacter sp. strain 638 and its host.
S. proteamaculans 568 is interesting, as it is, in contrast to
Enterobacter sp. strain 638, able to metabolize GABA and
PAA, two compounds involved in regulating plant responses to
stress. S. proteamaculans 568 is, like Enterobacter sp. strain 638,
able to produce 2-acetoin but lacks the acetoin reductase for
the bidirectional conversion of acetoin and diacetyl. Further-
more, it lacks the putative plant invasion functions found on
plasmid pENT628-1. A further comparison between both En-
terobacteriaceae strains should provide a better understanding
of the observed differences in their plant growth-stimulating
In the Enterobacteriaceae, sugar uptake dominantly occurs
via PTS systems, and both S. proteamaculans 568 and Entero-
bacter strain 638 contain PTS systems that are consistent with
their sugar metabolism (Table 1). S. proteamaculans 568 also
contains a copy of PTS from the glucitol family. The high
number of PTS genes found in the Enterobacteriales compared
to those found in the Pseudomonadales and the Xanthomonad-
ales is well known (4). Consistent with this observation is that
both P. putida W619 and S. maltophilia R551-3 possessed a
single gene coding for a PTS from the fructose family. This
PTS family is most prevalent in proteobacteria and is believed
to have evolved into other PTS systems (4).
P. putida W619 seems to be well adapted to influence the
phytohormone balances of its host: the strain appears to pro-
duce high levels of IAA and is able to metabolize PAA and
GABA. Elevated levels of GABA and PAA, a nonindolic auxin
that can account for up to one-half of the total bioassayable
auxin activity in plant extracts (45), can inhibit plant growth.
The complexity of the phytohormone balance points toward
the existence of a complex mechanism that fine-tunes the in-
teractions between P. putida W619 and other endophytes and
their poplar hosts. For instance, the negative effects on poplar
development observed after inoculation with M. populi BJ001
might reflect a disturbance of this balance, e.g., caused by
unnaturally high numbers of this bacterium during inoculation.
Many endophytic bacteria, such as S. maltophilia R551-3, are
closely related to pathogenic microorganisms whose genomes
have been or are in the process of being sequenced. Future
genome annotation and comparative genomics of endophytic
bacteria and phylogenetically closely related, nonendophytic
VOL. 75, 2009PLANT GROWTH-PROMOTING ENDOPHYTES FROM POPLAR TREES 755
microorganisms should result in the identification of the subset
of genes necessary for a successful endophytic colonization of
poplar trees. Understanding the interactions between endo-
phytic bacteria and their host plants, facilitated by the pub-
lished genome sequence of Populus trichocarpa (39), should
ultimately result in the design of strategies for improved poplar
biomass production as a feedstock for biofuels and bioreme-
This work was supported by the U.S. Department of Energy, Office
KP1102010, under contract DE-AC02-98CH10886. D.V.D.L. and S.T.
are also supported by Laboratory Directed Research and Develop-
ment funds from the Brookhaven National Laboratory under a con-
tract with the U.S. Department of Energy. This work was also funded
under Laboratory Directed Research and Development project
LDRD05-063. T.B. was supported by a postdoctorate grant from the
FWO-Flanders, Belgium. N.W. is presently supported by a Ph.D. grant
from IWT, Belgium.
We thank Bill Greenberg and Alina Sikar-Gang for technical assis-
tance and Dmytro Nykypanchuk (BNL CFN) for assisting with the
1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,
and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation
of protein database search programs. Nucleic Acids Res. 25:3389–3402.
2. Amann, R. I., W. Ludwig, and K.-H. Schleifer. 1995. Phylogenetic identifi-
cation and in situ detection of individual microbial cells without cultivation.
Microbiol. Rev. 59:143–169.
3. Arshad, M., and W. T. Frankenberger. 1991. Microbial production of plant
hormones. Plant Soil. 133:1–8.
4. Barabote, R. D., and M. H. Saier, Jr. 2005. Comparative genomic analyses of
the bacterial phosphotransferase system. Microbiol. Mol. Biol. Rev. 69:608–
5. Barac, T., S. Taghavi, B. Borremans, A. Provoost, L. Oeyen, J. V. Colpaert,
J. Vangronsveld, and D. van der Lelie. 2004. Engineered endophytic bacteria
improve phytoremediation of water-soluble, volatile, organic pollutants. Nat.
6. Betriu, C., A. Sanchez, M. L. Palau, M. Gomez, and J. J. Picazo. 2001.
Antibiotic resistance surveillance of Stenotrophomonas maltophilia, 1993–
1999. J. Antimicrob. Chemother. 48:152–154.
7. Bron, S., and G. Venema. 1972. Ultraviolet inactivation and excision-repair
in Bacillus subtilis. I. Construction and characterization of a transformable
eightfold auxotrophic strain and two ultraviolet-sensitive derivatives. Mutat.
8. Chen, X. H., A. Koumoutsi, R. Scholz, A. Eisenreich, K. Schneider, I. Hei-
nemeyer, B. Morgenstern, B. Voss, W. R. Hess, O. Reva, H. Junge, B. Voigt,
P. R. Jungblut, J. Vater, R. Sussmuth, H. Liesegang, A. Strittmatter, G.
Gottschalk, and R. Borriss. 2007. Comparative analysis of the complete
genome sequence of the plant growth-promoting bacterium Bacillus
amyloliquefaciens FZB42. Nat. Biotechnol. 25:1007–1014.
9. Do ¨bereiner, J., S. Urquiaga, and R. M. Boddey. 1995. Alternatives for ni-
trogen nutrition of crops in tropical agriculture. Fert. Res. 42:339–346.
10. Dylan, T., L. Ielpi, S. Stanfield, L. Kashyap, C. Douglas, M. Yanofsky, E.
Nester, D. R. Helinski, and G. Ditta. 1986. Rhizobium meliloti genes re-
quired for nodule development are related to chromosomal virulence genes
in Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. USA 83:4403–4407.
11. Germaine, K., E. Keogh, G. Garcia-Cabellos, B. Borremans, D. van der
Lelie, T. Barac, L. Oeyen, J. Vangronsveld, F. P. Moore, E. R. B. Moore,
C. D. Campbell, D. Ryan, and D. N. Dowling. 2004. Colonisation of poplar
trees by gfp expressing bacterial endophytes. FEMS Microbiol. Ecol. 48:109–
12. Germaine, K. J., X. M. Liu, G. G. Cabellos, J. P. Hogan, D. Ryan, and D. N.
Dowling. 2006. Bacterial endophyte-enhanced phytoremediation of the or-
ganochlorine herbicide 2,4-dichlorophenoxyacetic acid. FEMS Microbiol.
13. Glick, B. R. 2004. Bacterial ACC deaminase and the alleviation of plant
stress. Adv. Appl. Microbiol. 56:291–312.
14. Glick, B. R., C. B. Jacobson, M. K. Schwarze, and J. J. Pasternak. 1994.
1-Aminocyclopropane-1-carboxylic acid deaminase mutants of the plant
growth promoting rhizobacterium Pseudomonas putida GR 12-2 do not
stimulate canola root elongation. Can. J. Microbiol. 40:911–915.
15. Glick, B. R., D. M. Penrose, and J. Li. 1998. A model for the lowering of
plant ethylene concentrations by plant growth promoting bacteria. J. Theor.
16. Glick, B. R., B. Todorovic, J. Czarny, Z. Cheng, and J. Duan. 2007. Promo-
tion of plant growth by bacterial deaminase. Crit. Rev. Plant Sci. 26:227–242.
17. Glickmann, E., and Y. Dessaux. 1995. A critical examination of the speci-
ficity of the Salkowski reagent for indolic compounds produced by phyto-
pathogenic bacteria. Appl. Environ. Microbiol. 61:793–796.
18. Golubov, A., H. Neubauer, C. No ¨lting, J. Heesemann, and A. Rakin. 2004.
Structural organization of the pFra virulence-associated plasmid of rham-
nose-positive Yersinia pestis. Infect. Immun. 72:5613–5621.
19. James, K., and F. L. Olivares. 1997. Infection and colonization of sugar cane
and other Graminaceous plants by endophytic diazotrophs. Crit. Rev. Plant
20. Kuklinsky-Sobral, J., W. L. Araujo, R. Mendes, I. O. Geraldi, A. A. Pizzirani-
Kleiner, and J. L. Azevedo. 2004. Isolation and characterization of soybean-
associated bacteria and their potential for plant growth promotion. Environ.
21. Lodewyckx, C., J. Vangronsveld, F. Porteous, E. R. B. Moore, S. Taghavi, M.
Mergeay, and D. van der Lelie. 2002. Endophytic bacteria and their potential
applications. Crit. Rev. Plant Sci. 21:583–606.
22. Markowitz, V. M., F. Korzeniewski, K. Palaniappan, E. Szeto, G. Werner, A.
Padki, X. L. Zhao, I. Dubchak, P. Hugenholtz, I. Anderson, A. Lykidis, K.
Mavromatis, N. Ivanova, and N. C. Kyrpides. 2006. The integrated microbial
genomes (IMG) system. Nucleic Acids Res. 34:D344–D348.
23. Mastretta, C., T. Barac, J. Vangronsveld, L. Newman, S. Taghavi, and D. van
der Lelie. 2006. Endophytic bacteria and their potential application to im-
prove the phytoremediation of contaminated environments. Biotechnol.
Genet. Eng. Rev. 23:175–207.
24. Mayer, A. M. 1958. Determination of indole acetic acid by the Salkowsky
reaction. Nature 182:1670–1671.
25. Mergeay, M., D. Nies, H. G. Schlegel, J. Gerits, P. Charles, and F. Van
Gijsegem. 1985. Alcaligenes eutrophus CH34 is a facultative chemolithotroph
with plasmid-bound resistance to heavy metals. J. Bacteriol. 162:328–334.
26. Misaghi, I. J., and C. R. Donndelinger. 1990. Endophytic bacteria in symp-
tom free cotton plants. Phytopathology 80:808–811.
27. Monchy, S., M. A. Benotmane, R. Wattiez, S. van Aelst, V. Auquier, B.
Borremans, M. Mergeay, S. Taghavi, D. van der Lelie, and T. Vallaeys. 2006.
Proteomic analyses of the pMOL30 encoded copper resistance in Cupriavi-
dus metallidurans strain CH34. Microbiology 152:1765–1776.
28. Moore, F. P., T. Barac, B. Borremans, L. Oeyen, J. Vangronsveld, D. van der
Lelie, C. D. Campbell, and E. R. B. Moore. 2006. Endophytic bacterial
diversity in poplar trees growing on a BTEX-contaminated site: the charac-
terization of isolates with potential to enhance phytoremediation. Syst. Appl.
29. Ping, L. Y., and W. Boland. 2004. Signals from the underground: bacterial
volatiles promote growth in Arabidopsis. Trends Plant Sci. 9:263–266.
30. Ryu, C. M., M. A. Farag, C. H. Hu, M. S. Reddy, H. X. Wei, P. W. Pare, and
J. W. Kloepper. 2003. Bacterial volatiles promote growth in Arabidopsis.
Proc. Natl. Acad. Sci. USA 100:4927–4932.
31. Schatz, A., and C. Bovell, Jr. 1952. Growth and hydrogenase activity of a new
bacterium, Hydrogenomonas facilis. J. Bacteriol. 63:87–98.
32. Schlegel, H. G., H. Kaltwasser, and G. Gottschalk. 1961. Ein sumbersver-
fahren zur kultur wasserstoffoxidierender bacterien: wachstum physiolo-
gische untersuchungen. Arch. Mikrobiol. 38:205–222.
33. Shields, M. S., and M. J. Reagin. 1992. Selection of a Pseudomonas cepacia
strain constitutive for the degradation of trichloroethylene. Appl. Environ.
34. Shields, M. S., M. J. Reagin, R. R. Gerger, R. Campbell, and C. Somerville.
1995. TOM, a new aromatic degradative plasmid from Burkholderia (Pseudo-
monas) cepacia G4. Appl. Environ. Microbiol. 61:1352–1356.
35. Taghavi, S., H. Delanghe, C. Lodewyckx, M. Mergeay, and D. van der Lelie.
2001. Nickel-resistance-based minitransposons: new tools for genetic manip-
ulation of environmental bacteria. Appl. Environ. Microbiol. 67:1015–1019.
36. Taghavi, S., T. Barac, B. Greenberg, B. Borremans, J. Vangronsveld, and D.
van der Lelie. 2005. Horizontal gene transfer to endogenous endophytic
bacteria from poplar improves phytoremediation of toluene. Appl. Environ.
37. Tombolini, R., A. Unge, M. E. Davey, F. J. de Bruijn, and J. K. Jansson.
1997. Flow cytometric and microscopic analysis of GFP-tagged Pseudomo-
nas fluorescens bacteria. FEMS Microbiol. Ecol. 22:17–28.
38. Triplett, E. W. 1996. Diazotrophic endophytes: progress and prospects for
nitrogen fixation in monocots. Plant Soil 186:29–38.
39. Tuskan, G. A., S. DiFazio, S. Jansson, J. Bohlmann, I. Grigoriev, U. Hell-
sten, N. Putnam, S. Ralph, S. Rombauts, A. Salamov, J. Schein, L. Sterck, A.
Aerts, R. R. Bhalerao, R. P. Bhalerao, D. Blaudez, W. Boerjan, A. Brun, A.
Brunner, V. Busov, M. Campbell, J. Carlson, M. Chalot, J. Chapman, G. L.
Chen, D. Cooper, P. M. Coutinho, J. Couturier, S. Covert, Q. Cronk, R.
Cunningham, J. Davis, S. Degroeve, A. Dejardin, C. Depamphilis, J. Detter,
B. Dirks, I. Dubchak, S. Duplessis, J. Ehlting, B. Ellis, K. Gendler, D.
Goodstein, M. Gribskov, J. Grimwood, A. Groover, L. Gunter, B. Ham-
berger, B. Heinze, Y. Helariutta, B. Henrissat, D. Holligan, R. Holt, W.
Huang, N. Islam-Faridi, S. Jones, M. Jones-Rhoades, R. Jorgensen, C. Joshi,
J. Kangasjarvi, J. Karlsson, C. Kelleher, R. Kirkpatrick, M. Kirst, A.
Kohler, U. Kalluri, F. Larimer, J. Leebens-Mack, J. C. Leple, P. Locascio, Y.
756TAGHAVI ET AL.APPL. ENVIRON. MICROBIOL.
Lou, S. Lucas, F. Martin, B. Montanini, C. Napoli, D. R. Nelson, C. Nelson, Download full-text
K. Nieminen, O. Nilsson, V. Pereda, G. Peter, R. Philippe, G. Pilate, A.
Poliakov, J. Razumovskaya, P. Richardson, C. Rinaldi, K. Ritland, P. Rouze,
D. Ryaboy, J. Schmutz, J. Schrader, B. Segerman, H. Shin, A. Siddiqui, F.
Sterky, A. Terry, C. J. Tsai, E. Uberbacher, P. Unneberg, et al. 2006. The
genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science
40. Van Aken, B., C. M. Peres, S. L. Doty, J. M. Yoon, and J. L. Schnoor. 2004.
Methylobacterium populi sp. nov., a novel aerobic, pink-pigmented, facul-
tatively methylotrophic, methane-utilizing bacterium isolated from poplar
trees (Populus deltoides x nigra DN34). Int. J. Syst. Evol. Microbiol. 54:
41. Van Aken, B., J. M. Yoon, and J. L. Schnoor. 2004. Biodegradation of
nitro-substituted explosives 2,4,6-trinitrotoluene, hexahydro-1,3,5-trinitro-
1,3,5-triazine, and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine by a phyto-
symbiotic Methylobacterium sp. associated with poplar tissues (Populus del-
toides ? nigra DN34). Appl. Environ. Microbiol. 70:508–517.
42. van der Lelie, D., C. Lesaulnier, S. McCorkle, J. Geets, S. Taghavi, and J.
Dunn. 2006. Use of single-point genome signature tags as a universal tagging
method for microbial genome surveys. Appl. Environ. Microbiol. 72:2092–
43. Wang, C. X., E. Knill, B. R. Glick, and G. Defago. 2000. Effect of transferring
Pseudomonas fluorescens strain CHA0 and its gacA derivative CHA96 on
their growth-promoting and disease-suppressive capacities. Can. J. Micro-
44. Wang, Q., G. M. Garrity, J. M. Tiedje, and J. R. Cole. 2007. Naïve Bayesian
classifier for rapid assignment of rRNA sequences into the new bacterial
taxonomy. Appl. Environ. Microbiol. 73:5261–5267.
45. Wightman, F., and B. S. Rauthan. 1975. Evidence for the biosynthesis and
natural occurrence of the auxin, phenylacetic acid, in shoots of higher plants,
p. 15–27. In S. Tamura (ed.), Plant growth substances. Hirokawa Publishing,
Inc., Tokyo, Japan.
46. Worley, M. J., K. H. L. Ching, and F. Heffron. 2000. Salmonella SsrB
activates a global regulon of horizontally acquired genes. Mol. Microbiol.
acid (ACC)deaminase genesinto
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