R E S E A R C H A R T I C L E

Diversityof endophytic bacterial communities in poplargrownunder¢eld conditionsKristina Ulrich1, Andreas Ulrich2 & Dietrich Ewald1

1Bundesforschungsanstalt fur Forst- und Holzwirtschaft, Institut fur Forstgenetik- und Forstpflanzenzuchtung, Waldsieversdorf, Germany; and2Leibniz-Zentrum fur Agrarlandschaftsforschung (ZALF), Institut fur Landschaftsstoffdynamik, Muncheberg, Germany

Correspondence: Kristina Ulrich,

Bundesforschungsanstalt fur Forst- und

Holzwirtschaft, Institut fur Forstgenetik- und

Forstpflanzenzuchtung, Eberswalder

Chaussee 3a, D-15377 Waldsieversdorf,

Germany. Tel.: 149 33433 157 175; fax: 149

33433 157 199; e-mail:

k.ulrich@holz.uni-hamburg.de

Received 31 January 2007; revised 12 October

2007; accepted 16 October 2007.

First published online January 2008.

DOI:10.1111/j.1574-6941.2007.00419.x

Editor: Kornelia Smalla

Keywords

endophytic bacteria; bacterial community

composition; poplar; 16S rRNA gene.

Abstract

Bacterial endophytes may be important for plant health and other ecologically

relevant functions of poplar trees. The composition of endophytic bacteria

colonizing the aerial parts of poplar was studied using a multiphasic approach.

The terminal restriction fragment length polymorphism analysis of 16S rRNA

genes demonstrated the impact of different hybrid poplar clones on the endophytic

community structure. Detailed analysis of endophytic bacteria using cultivation

methods in combination with cloning of 16S rRNA genes amplified from plant

tissue revealed a high phylogenetic diversity of endophytic bacteria with a total of

53 taxa at the genus level that included Proteobacteria, Actinobacteria, Firmicutes

and Bacteroidetes. The community structure displayed clear differences in terms of

the presence and relative proportions of bacterial taxa between the four poplar

clones studied. The results showed that the genetic background of the hybrid

poplar clones corresponded well with the endophytic community structure. Out of

the 513 isolates and 209 clones identified, Actinobacteria, in particular the family

Microbacteriaceae, made up the largest fraction of the isolates, whereas the clone

library was dominated by Alpha- and Betaproteobacteria. The most abundant

genera among the isolates were Pseudomonas and Curtobacterium, while Sphingo-

monas prevailed among the clones.

Introduction

Endophytic bacteria reside within the inner parts of plant

hosts without causing disease symptoms and can be isolated

from surface-disinfested plants or extracted from internal

plant tissue (Hallmann et al., 1997). Various reports indi-

cated that endophytic bacteria exist in a variety of tissue

types within a broad range of plants, suggesting an ubiqui-

tous existence in nearly all higher plants. After penetration,

endophytes may colonize the plant systematically with

bacterial colonies and biofilms, residing latently in the

intercellular spaces, inside the vascular tissue or within cells

(Jacobs et al., 1985; Hurek et al., 1994; Di Fiori & Del Gallo,

1995). Although the interaction between endophytic bacter-

ia and their host plants is not fully understood, many

isolates showed beneficial effects on their hosts and may

play an important role in the physiology of the plant. Several

bacterial endophytes have been reported to support

plant growth by providing phytohormones, low-molecular

compounds or enzymes (Lambert & Joos, 1989; Frommel

et al., 1991; Glick et al., 1998). The colonization of ecological

niches is similar to that of phytopathogens. The release of

antimicrobial substances such as antibiotics or HCN (Ban-

gera & Thomashow, 1996; Blumer & Haas, 2000), the

production of siderophores (O’Sullivan & O’Gara, 1992) or

the induction of systemic resistance to pathogens (Liu et al.,

1995; Madhaiyan et al., 2004) favour them as candidates for

biological control. On the other hand, many well-known

plant pathogens could also be identified as typical endophy-

tic bacteria that cause no disease symptoms (Kobayashi &

Palumbo, 2000), but could become pathogenic under cer-

tain conditions or within different host genotypes (Misaghi

& Donndelinger, 1990).

Plants can be colonized simultaneously by a large variety

of endophytic bacteria. The variety of bacteria that has been

reported as endophytes spans a significant range of Gram-

positive and Gram-negative bacteria to include genera of

Alpha-, Beta- and Gammaproteobacteria, Actinobacteria,

FEMS Microbiol Ecol 63 (2008) 169–180 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Firmicutes and Bacteroidetes (Lodewyckx et al., 2002; Bacon

& Hinton, 2006). Most studies on the occurrence of

endophytic bacteria have been achieved using culture-de-

pendent approaches. However, due to the unknown growth

requirements of many bacteria and the presence of bacterial

cells that are in a viable but noncultivable state (Tholozan

et al., 1999), culture-dependent biodiversity studies of the

endophytic community are somewhat limited. Various

reports concerning endophytic bacteria in agricultural

plants demonstrated that the use of fingerprinting techni-

ques and clone analysis can provide additional information

for analysing the community composition of endophytic

bacteria (Chelius & Triplett, 2001; Garbeva et al., 2001;

Seghers et al., 2004; Sessitsch et al., 2004).

Although most of the studies concerning bacterial en-

dophytes were focused on agricultural and horticultural

plant species, endophytic bacteria were also detected in trees

like elm, pine, oak, citrus and coffee (Bacon & Mead, 1971;

Gardner et al., 1982; Araujo et al., 2002; Bent & Chanway,

2002; Mocali et al., 2003; Vega et al., 2005). For poplar trees,

which become more and more important not only as a

future energy source but also as ‘the trees of choice’ for

phytoremediation purposes and as target species for genetic

transformations (Van Aken et al., 2004b; Fladung & Ewald,

2006), most studies described only specific endophytic

strains (Germaine et al., 2004; Van Aken et al., 2004a; Doty

et al., 2005). In contrast, little is known about the commu-

nity composition of endophytic bacteria in poplar. Recently,

Moore et al. (2006) analysed the composition of culturable

endophytic bacteria in poplar grown on a benzene, toluene,

ethylbenzene and xylene (BTEX)-contaminated site and

reported a high diversity of isolates dominated by Gamma-

proteobacteria, especially Pseudomonas spp.

In this paper, the diversity of endophytic bacteria in aerial

parts of hybrid poplars grown in field trials under natural

conditions was systematically analysed using a combination

of culturing methods and direct amplification of 16S rRNA

genes from plant tissue. In addition, the community compo-

sition was compared with respect to different plant clones.

Materials and methods

Plant material and study sites

Samples were collected from four hybrid poplar clones

in summer 2005. The intersectional hybrid poplar clone

741 , [Populus alba L.� (Populus davidiana Dode1Populus

simonii Carr.)� Populus tomentosa Carr.] (Prof. Han, Beij-

ing, China) is derived from the species of two different

sections (Leuce and Tacamahaca) of the genus Populus,

while the hybrid clones Esch5 , (P. tremula L.�Populus

tremuloides Michx.), Brauna11 , (P. tremula L.) and W52 <(P. tremula L.) belong to the section Leuce. The plants

originated from cuttings from several trees and were grown

in experimental fields. The samples were obtained from 2-

year-old shoots in a clonal archive based on 10-year-old

stools that were cut down every second year. The clones 741

and Esch5/W were sampled at Waldsieversdorf, while the

clones Brauna 11, W52 and Esch5/G at Großhansdorf. The

field site Waldsieversdorf is characterized by a continental

climate with an average rainfall of 530 mm and nutrient-

poor sandy soils. In contrast, the Großhansdorf site has an

Atlantic climate with rainfalls of about 780 mm and is

located on a loamy sand.

Sample preparation

For terminal restriction fragment length polymorphism

(T-RFLP) analysis, 4 g of leaves and 4 g of branch sections

of about 2 cm (Ø = 5 mm) were sampled from four indivi-

dual trees as independent replicates for each plant clone. For

the screening of isolates and cloning, pooled samples from

five to 10 plants per poplar clone were used. The samples

were washed with tap water and surface-disinfected with

HgCl2. Leaves were disinfected in 0.1% HgCl2 containing a

drop of Tween 20 for 10 min. A higher concentration of

HgCl2 (0.25%) was used for branch sections. After three

rinses in sterile tap water, the bark of surface-disinfected

branches was removed with a sterilized razor blade, and the

branches were cut into 3–6 mm long pieces. To confirm that

the disinfection process was successful, the plant material

(pieces of the bark and leaves) was placed on nutrient agar

and the plates were examined for growth after incubation at

28 1C for 6 days. Nonsterile samples were discarded. The

samples were ground in 4 mL of sterile 0.85% NaCl with a

sterile mortar and pestle. Branch and leaf tissue extracts

were pooled to analyse the aerial endophytic bacterial

community.

T-RFLP

Bacterial pellets from ground plant material were obtained

by differential centrifugation and stored at � 80 1C. The

pellets were homogenized in Eppendorf tubes containing a

lysing matrix (Fast DNA Spin Kit for Soil; QBIOgene,

Carlsbad), using a shaking mill (Retsch GmbH, Haan,

Germany) for 4 min at maximum speed and the total DNA

was extracted following a miniaturized CTAB-based proto-

col (Dumolin et al., 1995). The bacterial primers 799f

(Chelius & Triplett, 2001) labelled at the 50-end with

6-carboxyfluorescein (6-FAM) and 1525r (Lane, 1991) were

used to amplify a fragment of c. 760 bp of the 16S rRNA

gene. PCR reaction (50mL) was performed using AccuPrime

polymerase with buffer II (Invitrogen, Carlsbad) to which

10 ng of template DNA were added. Thermal cycling condi-

tions were: 2 min denaturation at 94 1C followed by 35 cycles

of 20 s at 94 1C, 40 s at 53 1C and 60 s at 68 1C, and a final

FEMS Microbiol Ecol 63 (2008) 169–180c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

170 K. Ulrich et al.

extension for 7 min at 68 1C. The PCR products were

examined in a 1% agarose gel and subsequently purified

with an Invisorbs Spin PCRapid Kit (Invitek, Berlin,

Germany). T-RFLP analysis was performed as described by

Ulrich & Becker (2006) with HhaI-digested PCR products.

The samples were separated with GeneScan 1000 Rox

(Applied Biosystems) as an internal size standard on an

ABI 310 DNA sequencer (Applied Biosystems) using POP6

polymer. Data analysis of the T-RFLP profiles was per-

formed using the GELCOMPAR II software v. 2.5 (Applied

Maths, Saint-Martens-Latem, Belgium) and the ABI files

were converted into the GELCOMPAR curve format. To consider

both the presence and the relative abundance of terminal

restriction fragments (T-RFs), densitometric curves of

whole profiles were analysed through Pearson’s correlation.

The resulting similarity matrix was the basis for clustering

by the Ward algorithm (Ward, 1963). T-RFLP profiles in the

range of 35–740 bp were used for the cluster analysis. To test

for significant differences between T-RFLP profiles, the

Pearson’s similarity matrix was used for a permutation test

as described by Smalla et al. (2007). The significance of

effects was indicated by the difference of mean within-group

and between-group similarities (Kropf et al., 2004).

Screening of isolates

For the isolation of endophytic bacteria, ground tissue

extracts were serially diluted in 0.85% NaCl and plated on

R2A, a nutrient-poor medium suitable for the growth of

diverse plant-associated bacteria (Difco, Detroit) to recover

bacterial endophytes. Plates were incubated for 3–6 days at

28 1C. CFU were counted and expressed as CFU per gram of

fresh weight. In total, at least 80 bacterial colonies per poplar

clone were randomly selected and subcultured on R2A or

tryptic soy agar (TSA, Sigma-Aldrich Co., St Louis).

Identification of endophytic bacteria

For DNA extraction, single colonies were resuspended in

20 mL 25 mM NaOH/0.25% sodium dodecyl sulphate and

heated for 15 min at 95 1C. Aliquots (0.1mL) of the resulting

lysate were directly used for PCR without further purifica-

tion. The 16S rRNA gene fragments of the bacterial isolates

were amplified using primers 799f and 1525r. For routine

assays, a 50 mL reaction mixture containing 2.5 U Taq-

Polymerase (AppliChem, Darmstadt, Germany), 2.5 mM

MgCl2, 200 mM of each dNTP and 15 pmol of each primer,

was used. The amplifications were performed with the

following protocol: initial denaturation at 95 1C for 3 min;

25 cycles of 30 s at 94 1C, 40 s at 53 1C, 1.5 min at 72 1C,

followed by a single final extension at 72 1C for 8 min and

a final soak at 4 1C. PCR products were examined electro-

phoretically in a 1% agarose gel. The PCR products were

purified with an Invisorbs Spin PCRapid Kit (Invitek,

Berlin, Germany). DNA sequencing was performed using a

BigDye terminator cycle sequencing Kit (Applied Biosys-

tems, Foster City) and the primer 1492r. As suggested by

Stackebrandt & Rainey (1996), single strand sequencing of

about 400–600 bp was applied, which was sufficient for a

precise taxonomic placement of the 513 isolates in commu-

nity analysis. The assignment of the isolates at genus or

family level was based on the BLAST analysis and on the

taxonomic assignment by the CLASSIFIER program of the RDP.

The composition of the identified genera of culturable

endophytic bacteria was analysed by UPGMA clustering

using the percent disagreement method (STATISTICA 6.1,

StatSoft, Tulsa). For phylogenetic analysis, 30 isolates (one

for each identified taxum) were chosen for sequencing both

complementary strands with the primers 1492r and 799f.

Cloning of bacterial 16S rRNA genes

Isolation of total DNA from the endophytic community of

the poplar clones Esch5/W and 741 was performed as

described for T-RFLP analysis. The 16S rRNA gene frag-

ments were amplified using the same PCR primers as above,

purified using the Invisorbs Spin PCRapid Kit (Invitek)

and cloned into pCR2.1 (TA Clonings Kit, Invitrogen,

Carlsbad). About 100 positive clones per sample were

selected. Sequencing was performed as described for the

isolates. 209 clones were identified by single strand sequen-

cing of about 400–600 bp and 51 clones were chosen for

phylogenetic analysis. The partial 16S rRNA gene sequences

were evaluated by the program CHIMERA CHECK of the

Ribosomal Database Project (RDP, http://rdp8.cme.msu.

edu/cgis/chimera.cgi?su=SSU; Cole et al., 2005) to detect

chimeric artefacts. Clones harbouring a sequence with a

potential chimeric structure were excluded from further

analyses.

Phylogenetic analysis

The DNA sequences of representative isolates and clones, as

well as various 16S rRNA gene sequences for selected

reference strains were aligned by the CLUSTALW algorithm

(programme version 1.74; Thompson et al., 1994). The

phylogenetic trees were constructed using the Neighbor-

Joining and Maximum-Likelihood algorithms (PHYLIP com-

puter program package, version 3.6a2; Felsenstein, 1993).

The Neighbor-Joining algorithm (Saitou & Nei, 1987) was

based on a matrix of pair-wise distances corrected for

multiple base substitutions (Kimura, 1980) with a transi-

tion/transversion ratio of 2.0. The Maximum-Likelihood

method (Felsenstein, 1981) was applied with three jumbles

of the data set (randomized input order) and without global

rearrangement. The unrooted trees were calculated using the

16S rRNA gene sequence of Escherichia coli (J01695) or

Microbacterium lacticum (D21343) as an outgroup. The 16S

FEMS Microbiol Ecol 63 (2008) 169–180 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

171Endophytic bacteria in poplar

rRNA gene sequences obtained in this study (one isolate or

clone per taxum identified) were deposited in the EMBL

nucleotide sequence database under the accession nos.

AM489612–AM489640, AM489641–AM489691 and

AM698087.

Results

Molecular fingerprinting of endophyticbacterial communities

T-RFLP analysis of 16S rRNA genes resulted in comparable

profiles for the aerial endophytic bacterial community of the

four poplar clones studied. The profiles showed eight to 14

dominant T-RFs and several small T-RFs. The profiles of the

individual trees of poplar clone 741 displayed the highest

number of T-RFs and two dominant T-RF’s (440 and

664 bp), which were not identified in the profiles of the

endophytic community of the other poplar clones. As a

result of analysing the densitometric curves of the profiles,

the similarity of the bacterial communities is shown in

Fig. 1. The four replicates (individual trees) of three poplar

clones formed separate branches. In the case of Esch5/W,

three replicates clustered together. The test showed signifi-

cant results in the global comparison (Po 0.001) as well as

in all pair-wise comparisons (P = 0.029) except for the

comparison of Esch5 with Brauna11 and with 741. This

demonstrated a higher similarity within a poplar clone than

between the different poplar clones. The first branch of the

dendrogram clearly differentiates the intersectional hybrid

poplar 741 from the other clones indicating the most

evident differences in the community structure of that

poplar clone.

Culturable endophytic bacteria

To describe the diversity of endophytic bacteria in poplar, as

well as differences between the poplar clones in more detail,

culturable bacteria from pooled samples were analysed

from hybrid poplar clones (Esch5 was sampled from

two locations). Population densities of endophytic bacteria

recovered from R2A medium ranged from 8� 104 to

6� 105 CFU g�1 fresh weight. A total of 513 bacterial isolates

was identified at the genus level by partial sequencing of the

16S rRNA genes. The endophytic isolates obtained from

all poplar clones could be assigned to 27 different genera

(in one case to unclassified Microbacteriaceae). The majority

of isolates were related to common plant-associated,

endophytic or soil bacteria. Actinobacteria dominated the

collection of isolates, comprising 49% of the total isolates,

which includes Microbacteriaceae (47% of total isolates),

Sanguibacteriaceae (2%) and one isolate of Nocardiaceae.

The genera Curtobacterium (19%) and Plantibacter (9%)

represented the majority of the Microbacteriaceae. Gamma-

proteobacteria were the second most abundant class (28%

of total isolates) dominated by Pseudomonas (19%) and

Xanthomonas (7%) species. Alphaproteobacteria comprised

17% of the total isolates, represented largely by Sphingomo-

nas spp. (10%) and Methylobacterium spp. (5%). Bacteria

belonging to Bacteroidetes were represented mainly by

the genus Pedobacter and formed only 4% of the total

isolates. Firmicutes (Paenibacillus) comprised only 1.4% of

the total isolates.

The composition of the culturable endophytic bacteria

found in the single poplar clones is demonstrated in Fig. 2a.

The poplar clones showed clear differences in terms of the

relative proportions of Alpha- and Gammaproteobacteria as

well as Actinobacteria. Bacteroidetes isolates could only be

found in the Brauna11 and W52 clones. The seven isolates

belonging to Firmicutes were obtained from W52. Moreover,

differences in presence and relative proportions of the

identified genera were obvious between the poplar clones

(Table 1). The poplar clone 741 showed the highest diversity

with 17 identified genera, followed by Esch5/G and W52

with 14 and 12 genera. Esch5/W and Brauna11 comprised

10 and 9 genera, respectively. In Esch5 from both sites, the

same genera of Microbacteriaceae could be detected and the

composition of Gammaproteobacteria was similar, too.

The poplar clones Brauna11 and W52 displayed similarities

in their endophytic isolates with high proportions of Pseu-

domonas (33% and 46%) and Curtobacterium (40% and

28%). Moreover, isolates belonging to the genera Chryseo-

bacterium and Pedobacter could only be obtained from the

P. tremula clones Brauna11 and W52. The similarity of the

Fig. 1. Dendrogram showing the similarity between the T-RFLP profiles

of the endophytic bacterial community derived from the four hybrid

poplar clones (four replicates).

FEMS Microbiol Ecol 63 (2008) 169–180c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

172 K. Ulrich et al.

culturable bacterial communities from the poplar clones was

analysed by UPGMA clustering. In correspondence to the T-

RFLP analysis, the composition of the isolates of poplar

clone 741 was distinguished from the other plant clones

(data not shown). The cluster analysis also demonstrated the

similarity of the endophytic isolates from the poplar clone

Esch5 grown at two locations and the similarity of the

isolates from Brauna11 and W52.

Endophytic bacterial communities analysedusing a clone library

Cloning was performed for poplar clones 741 and Esch5

from the location Waldsieversdorf. The total of 209 clones

analysed resulted in 37 different taxa at the genus level. The

majority of clones were assigned to Proteobacteria (82%)

with Alphaproteobacteria as the predominant group (51% of

total clones). Most of these clones belonged to Sphingomo-

nas (36% of total clones), which corresponded with the high

abundance of Sphingomonas among the Alphaproteobacteria

isolates. Betaproteobacteria were the second most abundant

class (27% of total clones) represented mainly by Oxalobac-

teraceae. This is in contrast to the analysis of the culturable

bacteria, where no betaproteobacterial isolates could be

obtained. The occurrence of Actinobacteria in the clone

library was only 8.5% compared with an abundance of 49%

within the isolates. The remainder of the clone library is

comprised of members of Gammaproteobacteria, Bacteroi-

detes, Firmicutes and unclassified bacteria. The phylogenetic

overlap between genera obtained through cultivation meth-

ods and found by direct PCR amplification of the 16S rRNA

genes was 21%. The isolate collection comprised 51% and

the clone library 70% of the total number of genera found.

The comparison of endophytic bacterial communities

obtained from poplar clones 741 and Esch5/W is shown in

Table 2 and Fig. 2b. Twenty-seven different taxa at the genus

level could be found in 741, while only 19 taxa were detected

in Esch5, which corresponded to the highest diversity of the

T-RFLP profiles and of the isolate collection for the poplar

clone 741. For both poplar clones, Alphaproteobacteria were

the most abundant group, followed by Betaproteobacteria.

Against the backdrop of low Actinobacteria proportion in

the total clones, 15% of the clones from 741 could be

assigned to Actinobacteria, which is in accordance with the

high percentage of Actinobacteria among culturable bacteria

isolated from this poplar. In contrast, only 2% of the total

clones were Actinobacteria in Esch5. The amount of Gam-

maproteobacteria and Bacteroidetes clones was comparable

in both poplars.

Analysing of the 16S rRNA gene sequences resulted in 11

theoretical T-RFs for the clone library which corresponded

well with the presence of the respective peaks and their

relative abundances in the T-RFLP profiles. Thus, the 78 and

329 bp T-RFs which were represented in the clone library

by Sphingomonas and Pseudomonas, and by genera of

Betaproteobacteria, Gammaproteobacteria and Firmicutes,

respectively, were most predominant among the clones and

in the T-RFLP profiles and abundant among the isolates as

well. Furthermore, the abundant T-RF of 192 bp was only

found in the clone library and the T-RFLP analysis, whereas

an abundant T-RF (45 bp, represented mainly by actinobac-

terial genera) of the isolates was inconsiderable in both the

clone library and the T-RFLP analysis.

Phylogenetic analysis of isolates and clones

The relatedness of representative isolates and clones (one for

each taxum found) is shown in supplementary Tables S1 and

S2. Most of the isolates and the majority of clones showed a

similarity of above 99% to known reference strains. In some

cases, clearly distinct groups within one genus were ob-

tained. Six clones could not be assigned to any bacterial taxa

(a) (b)

Fig. 2. Distribution of the bacterial classes of

endophytic bacteria obtained from the hybrid

poplar clones by cultivation methods (a) and

cloning of 16S rRNA genes amplified directly

from plant material (b). Isolates were screened

from four different plant clones with clone

Esch5 sampled at two locations. A clone library

was studied for the clones 741 and Esch5 grown

at Waldsieversdorf. The number of isolates and

clones, respectively, studied for each plant clone

is given in brackets.

FEMS Microbiol Ecol 63 (2008) 169–180 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

173Endophytic bacteria in poplar

based on the 16S rRNA gene sequence similarity. Five of

these clones, represented by Esch5-32 in supplementary

Table S2, were closely related with an uncultured bacterium

clone from the cryptoendolithic communities from McMur-

do Dry Valleys of Antarctica (De la Torre et al., 2003). The

clone Esch5-52 showed a strong similarity to an unclassified

clone obtained from volcanic deposits in Hawaii (supple-

mentary Table S2).

The phylogenetic relationship of isolates and clones in

comparison to the type strains of related species is demon-

strated for three selected bacterial classes (Fig. 3). The

Actinobacteria as the most abundant group of isolates

displayed a high diversity of phylogenetically related genera.

Actinobacterial isolates often showed a close relationship to

the species of the family Microbacteriaceae, while the clones

belonged to species of other families of Actinobacteria, as

well as Microbacteriaceae (Fig. 3a). Thus, more families of

the Actinobacteria could be found by cloning, even though

only a minor part of the total clones was assigned to

Table 1. Composition of endophytic isolates obtained from the four

hybrid poplar clones

Identified taxum

Waldsieversdorf� Großhansdorf

741 Esch5/W Esch5/G Brauna11 W52

Alphaproteobacteria

Agrobacterium 3w

Rhizobium 7 2 1

Methylobacterium 9 7 6 1

Roseomonas 1

Sphingomonas 32 14 2 3

Gammaproteobacteria

Enterobacter 1

Erwinia 1 1

Pantoea 3

Pseudomonas 1 23 3 34 38

Luteibacter 1

Stenotrophomonas 3 1

Xanthomonas 2 15 19

Actinobacteria

Curtobacterium 3 19 10 41 23

Clavibacter 5 1 17 3

Plantibacter 39 3 1 1

Okibacterium 18

Rathayibacter 4 11

Agreia 1

Microbacterium 17 3 1

Frigoribacterium 1 5 4

Cryocola 5

Unclassified

Microbacteriaceae

2 2 1

Sanguibacter 11

Nocardioides 1

Firmicutes

Paenibacillus 7

Bacteroidetes

Chryseobacterium 2 3

Pedobacter 12 2

Total isolates 155 94 80 102 82

�The clones 741 and Esch5/W were sampled at Waldsieversdorf, while

the clones Brauna 11, W52 and Esch5/G at Großhansdorf.wThe number of isolates obtained from the respective poplar clone is

presented for each identified taxum.

Table 2. Composition of endophytic bacteria obtained from two hybrid

poplar clones by cloning of 16S rRNA genes amplified directly from plant

material

Identified taxum 741� Esch5/W

Alphaproteobacteria

Caulobacter 3w

Brevundimonas 1

Sphingomonas 43 32

Sphingobium 2

Methylobacterium 5 9

Rhizobium 2

Acetobacteraceae 2

Aurantimonadaceae 7

Betaproteobacteria

Acidovorax 1 1

Variovorax 4 1

Comamonadaceae 3

Oxalobacter 10

Massilia 5 3

Oxalobacteraceae 3 14

Xylophilus 3

Leptothrix 1

Burkholderiales genera incertae sedis 5 1 2

Alcaligenaceae 1

Burkholderia 3

Neisseriales 1

Gammaproteobacteria

Pseudomonas 3 2

Xanthomonas 2

Xanthomonadaceae 2

Actinobacteria

Frigoribacterium 8 2

Curtobacterium 1

Clavibacter 1

Microbacterium 2

Beutenbergiaceae 1

Kineococcus 1

Frankineae 1

Friedmanniella 1

Firmicutes

Anaerococcus 1

Bacteroidetes

Flexibacteraceae 2

Chryseobacterium 2

Pedobacter 4

Sphingobacteriales 4

Unclassified Bacteria 6

Total clones 110 99

�The clones 741 and Esch5 were grown at Waldsieversdorf.wThe number of clones obtained from the respective poplar clone is

presented for each identified taxum.

FEMS Microbiol Ecol 63 (2008) 169–180c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

174 K. Ulrich et al.

Actinobacteria. As displayed in the phylogenetic tree, endo-

phytic bacteria related to Kineococcus, Solicoccus, Salana, and

Friedmanniella were only found in the clone library.

Alpha- and Gammaproteobacteria contained isolates or

clones belonging to distinct groups of the same genus.

Within Sphingomonas, which was found to be one of the

most abundant genera, isolates and clones related to several

species could be revealed (Fig. 3b). In total, endophytic

isolates and clones belonging to at least seven different

species of sphingomonads were found. Similar results were

obtained for the genus Pseudomonas with close relationships

to Pseudomonas trivialis, Pseudomonas lutea, Pseudomonas

syringae and Pseudomonas rhizosphaerae. Several clones

showed an identical sequence to Xanthomonas populi, which

is known as the cause of bacterial stem canker of poplar

(Ride, 1993). However, other Xanthomonas species like

Xanthomonas campestris and Xanthomonas gardneri also

had the same 16S rRNA gene sequences.

Discussion

The objective of this study was to examine the community

composition of endophytic bacteria from different poplar

clones growing under field conditions. A high phylogenetic

diversity of endophytic bacteria could be revealed with a

total of 53 taxa at the genus level that included Proteobacter-

ia, Actinobacteria, Firmicutes and Bacteroidetes. Only little

is known about the endophytic bacterial community har-

boured by poplar plants from other studies. Recently, Moore

et al. (2006) described the diversity of endophytic bacteria in

two cultivars of Populus trichocarpa� Populus deltoides

grown in a phytoremediation field trial on a site contami-

nated with BTEX compounds. High numbers of Gamma-

proteobacteria (61%) were found in the isolate collection,

which was dominated by Pseudomonas spp. that reached up

to 53% of the total isolates in one cultivar. In contrast,

Gammaproteobacteria made up only 28% of the total isolates

and 4% of the clones, with Pseudomonas species comprising

19% of the isolates and only 2% of clones in this study. The

highest percentage of Pseudomonas in a single poplar clone

was found in the W52 with about 46%. The abundance of

Pseudomonas within endophytic bacteria was found to be

highly variable and seems to be dependent on the plant

clone or environmental conditions. The high abundance of

Pseudomonas isolates in poplar grown in the phytoremedia-

tion field may be due to their relatively high amount in the

C

C

C

C

C

C

C

C

I

(a)

Fig. 3. Phylogenetic trees showing the rela-

tionship of the 16S rRNA gene sequences of

representative isolates and clones belonging to

Actinobacteria (a), as well as Alpha- and Gam-

maproteobacteria (b). The trees were generated

using the Neighbor-Joining algorithm. Branches

also found by the Maximum-Likelihood method

are indicated by dots. Type strains of each of the

shown species are used for the calculation. The

16S rRNA gene sequence of Erwinia billingiae

Eb661 (AM055711) was used instead of that of

the type strain (Y13249) to improve sequence

quality. Scale bar = 0.05 relative sequence diver-

gences.

FEMS Microbiol Ecol 63 (2008) 169–180 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

175Endophytic bacteria in poplar

contaminated soil. In this context, Pseudomonas strains have

been reported to be involved in the biodegradation of

2.5-dichlorobenzoate, toluene and naphthalene (Simon

et al., 1993; Crowley et al., 1996; Parales et al., 1996; James

& Williams, 1998). In correspondence to the results found

for poplar, a study by Siciliano et al. (2001) indicated an

enrichment of bacterial endophytes containing specific

catabolic genotypes in different grasses dependent on the

presence of contaminants and suggested that environmental

impacts play an important role in controlling or altering the

composition of the endophytic microbial community. Be-

sides this, plants may have the ability to selectively enhance

C

C

C

C

CC

C

C

C

C

CC

CC

C

C

C

C

C

C

(b)

Fig. 3. continued

FEMS Microbiol Ecol 63 (2008) 169–180c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

176 K. Ulrich et al.

the prevalence of endophytes containing specific catabolic

genes or other properties that benefit plant growth under

specific environmental conditions. Thus, the finding of a

different endophytic community in poplars grown under

normal field conditions and in BTEX-polluted soils pro-

vided further evidence that the mechanism by which plants

control their endophytic bacteria and/or environmental

conditions strongly influence the diversity and composition

of the endophytic bacterial community.

Several studies on agricultural plants revealed the endo-

phytic community as a subset of the soil community

(Germida et al., 1998; Berg et al., 2005; Gotz et al., 2006).

This is also suggested by the results of this study, because

several phylogenetically diverse bacteria, which are also

known as common soil bacteria could be found. On the

other hand, various species found in poplar are typical

phyllosphere bacteria, for example Plantibacter and Curto-

bacterium (Behrendt et al., 2002; Beattie, 2006). Thus,

penetration of the host plant via leaf stomata and stem

lenticels (Kluepfel, 1993), as well as through folia damages

(Leben et al., 1968) may also play an important role.

The T-RFLP analysis revealed significant differences be-

tween the studied poplar clones, which demonstrated the

impact of the plant clone on the endophytic community

structure. All approaches used in this study showed consis-

tently strong differences, as well as the highest diversity of

endophytic bacteria for the intersectional Chinese hybrid

poplar 741 derived from four different Populus species. On

the other hand, the P. tremula clones W52 and Brauna11

displayed a high similarity in their endophytic isolates.

Thus, the genetic background of the hybrid poplar clones

corresponded well with the endophytic community struc-

ture. The impact of plant clones was also observed for

various tree species by other authors. Cambours et al.

(2005) found differences in the endophytic pathogenic and

ice nucleation active bacteria in four Salix clones. The

diversity of endophytic bacteria was also influenced by

different plant genotypes of citrus and poplar (Araujo

et al., 2002; Moore et al., 2006). Thus, endophytic bacterial

communities seem to be dependent on the genotype of

long-living plants, suggesting that each tree species or clone

has an association to specific bacterial endophytes.

Cultivation as well as culture-independent methods were

used to analyse the endophytic community composition of

poplar clones. Both approaches revealed differences between

the poplar clones in terms of the diversity and abundance of

their bacterial classes and genera, whereas the culturable

component of the bacterial community was different from

that obtained by clone analysis. While Actinobacteria, espe-

cially the family Microbacteriaceae represented the majority

of the isolates, the clone library was dominated by Alpha-

proteobacteria and Betaproteobacteria. Some Actinobacteria

are recalcitrant to cell lysis and therefore, they might be

underrepresented in a clone library. However, the selectivity

of cultivation as well as a preferential amplification of

certain bacterial groups with the eubacterial primers could

also cause the different abundance. A disparity in the

representation of different bacterial classes, genera and

species between isolate collection and clone library had also

been observed in several other studies (Dunbar et al., 1999;

Hengstmann et al., 1999; Chelius & Triplett, 2001; Idris

et al., 2004). Therefore, the combination of culturing

methods and cloning analysis is needed for the study of the

endophytic communities of woody plants.

Curtobacterium strains have been isolated as typical

endophytes from several woody plants like sweet-orange,

coffee and grapevine (Bell et al., 1995; Araujo et al., 2002;

Vega et al., 2005). In this study, Curtobacterium could be

obtained from all poplar clones and comprised 19% of total

isolates, whereas the majority were assigned to the species

Curtobacterium flaccumfaciens. Several subspecies or strains

of C. flaccumfaciens are known as causal agents of wilt and

necrotic symptoms on horticultural and ornamental plants

(Vidaver, 1982). However, C. flaccumfaciens had also been

reported as a biocontrol agent for cucumber (Raupach &

Kloepper, 2000) and to play a role in triggering induced

systemic resistance (Raupach & Kloepper, 1998). In citrus, a

higher frequency of C. flaccumfaciens was found in asymp-

tomatic plants compared with plants infected with Xylella

fastidiosa, further suggesting the role of this bacterium in the

resistance of plants to chlorosis (Araujo et al., 2002). In this

context, it remains open whether identical Curtobacterium

strains act as pathogens or antagonistic strains in depen-

dence on the genetic background of the host plant and

environmental conditions, or if different strains of the same

species mediate such different properties.

Some isolates and clones were highly similar to Clavibac-

ter michiganensis, Pseudomonas syringae and Xanthomonas

populi, which have been historically thought to be patho-

genic (Lindow et al., 1978; Shirakawa et al., 1991; Ride, 1993;

Ramstedt et al., 1994). However, no visible symptoms of

disease had been detected on the poplar plants analysed.

In both poplars studied by cloning of 16S rRNA genes, the

highest abundance at the genus level could be found for

Sphingomonas. Among the culturable bacteria, varying

proportions (up to 21%) were detected from the studied

poplars. The high abundance found in some of the poplar

clones combined with the high phylogenetic variability of

sphingomonads suggests a putative function within the

bacterial endophytes.

Besides the discussed relation of endophytic and patho-

genic bacteria, bacterial endophytes may also be important

in forest ecosystems for increasing the phenotypic plasticity

of their long-living tree hosts under variable or deleterious

environmental conditions, e.g. during periods of drought or

nutrient deprivation (Chanway, 1998). However, in contrast

FEMS Microbiol Ecol 63 (2008) 169–180 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

177Endophytic bacteria in poplar

to agricultural plants, where many effects had been de-

scribed (Bacon & Hinton, 2006), little was known about

their interaction and influence on plant growth until now.

There is only some evidence of plant growth-promoting

activities by endophytic bacteria in trees, such as, stimula-

tory effects on seedling growth caused by endophytic

pseudomonads, Bacillus strains and actinomycetes (Gardner

et al., 1984; Chanway & Holl, 1994). However, further

studies are needed to ascertain the association between

woody plants and the wide range of different bacteria living

there as endophytes.

In conclusion, this study revealed a high phylogenetic

diversity of bacterial endophytes in poplar and showed an

influence of the plant genotype on the endophytic bacterial

community. Knowledge on the abundance and composition

of bacterial endophytes is an indispensable precondition for

future applications in areas such as plant health promotion,

phytoremediation, and studies on horizontal gene transfer

between endophytic bacteria and during Agrobacterium-

mediated plant transformation.

Acknowledgements

This work was supported by grant 0313285I from the

Federal Ministry of Education and Research. The authors

are grateful to Mrs Hannelore Enkisch and Mrs Sigune

Weinert for their excellent technical assistance, and to Dr

Siegfried Kropf for helping with the statistical analyses. The

authors also thank Prof. Yang from the Agricultural Uni-

versity Hebei, Baoding for providing the poplar clone 741

and Dr Matthias Fladung for providing plant material from

poplar clones.

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Supplementarymaterial

The following supplementary material is available for this

article online:

Table S1. 16S rRNA gene sequence similarities of

representative endophytic bacterial isolates from the identi-

fied taxa.

Table S2. 16S rRNA gene sequence similarities of

representative bacterial clones of the identified taxa ob-

tained from endophytic communities of poplar clones 741

and Esch5/W.

This material is available as part of the online article

from: http://www.blackwell-synergy.com/doi/abs/10.1111/

j.1574-6941.2007.00419.x (This link will take you to the

article abstract).

Please note: Blackwell Publishing is not responsible for

the content or functionality of any supplementary materials

supplied by the authors. Any queries (other than missing

material) should be directed to the corresponding author for

the article.

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180 K. Ulrich et al.