Plant Cell Tiss Organ Cult (2008) 93:347–351

DOI 10.1007/s11240-008-9367-z

RESEARCH NOTE

Paenibacillus—a predominant endophytic bacterium

colonising tissue cultures of woody plants
Kristina Ulrich Æ Thomas Stauber Æ Dietrich Ewald

Received: 29 November 2007 / Accepted: 8 March 2008 / Published online: 26 March 2008
Ó Springer Science+Business Media B.V. 2008

Abstract High densities of endophytic bacteria Bacterial contamination is a serious problem in plant
were found in plant material from poplar, larch and tissue cultures of trees. Bacteria that may be intro-
spruce that had been micropropagated for at least duced in cultures as endophytes or later during
5 years. The majority of these bacteria were assigned culture handling sometimes remain covert or latent
to the genus Paenibacillus based on the sequencing of with no obvious growth on tissue culture medium
the 16S rRNA genes. Other endophytic bacteria such (Ewald et al. 2000). They may affect culture growth
as Methylobacterium, Stenotrophomonas or Bacillus under the modified conditions in vitro and act as
could also be found but only in some tissue cultures. ‘‘vitropathogens’’ (Leifert and Cassells 2001). Some
Certain species or strains of Paenibacillus, especially endophytes are otherwise known to enhance plant
those with a close relationship to P. humicus, seemed growth through the production of hormones, enzymes
to accumulate under in vitro conditions without or antibiotic compounds (Holland and Polacco 1994).
visible negative influences on the plant’s develop- In contrast to visibly growing bacterial contaminants,
ment. Poplar microcuttings inoculated with the the effect of covert bacteria is difficult to assess and
endophytic Paenibacillus isolate 22 showed signifi- the activities of endophytes often pretend plant
cantly more roots per cutting and higher root length activities. In this study, culturable endophytic bacte-
in comparison to the control plants after 3 weeks. ria were systematically investigated in in vitro
propagated tissue cultures of different tree species
Keywords Micropropagation
Poplar
Root of different origins.
growth promotion Five hybrid poplar clones, a triploid hybrid of
Chinese white poplar (clone 73), and eight transgenic
lines obtained from the poplar clone 741 carrying
Abbreviations genes for insect resistance were included in this study.
Cfu Colony forming units Furthermore, three hybrid larch clones, one Norway
spruce clone and two clones from black locust were
also tested. The in vitro plant clones were initiated
from surface-disinfected shoot tips (poplar: Yang
K. Ulrich (&)
T. Stauber
D. Ewald et al. 2006; Fladung et al. 1997; black locust: Naujoks
Federal Research Centre for Forestry and Forest Products, et al. 2000); immature or mature zygotic embryos
Institute for Forest Genetics and Forest Tree Breeding,
(SELA 30/4: Ewald and Hu 2003; Marienberg: Ewald
Eberswalder Chaussee 3A, Waldsieversdorf 15377,
Germany and Hu 2007); seedlings (HL 86/6: Schneck and
e-mail: k.ulrich@holz.uni-hamburg.de Ewald 2001) or winter buds (adult hybrid larch LSP2:

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Ewald 2007). The plant clones were established in The isolation of endophytic bacteria was performed
different laboratories (Table 1). All clone lines were as described by Ulrich et al. (2008). Colony forming
sub-cultured for at least 5 years. Cultures were units were counted and expressed as cfu per gram of
incubated at 22°C under 16 h of photoperiod fresh weight. Only colonies with identical morphol-
(28 lmol m-2 s-1 for broadleaved species and con- ogy could be found. Eight colonies per plant clone
tinuous red light at 30–35 lmol m-2 s-1 for conifers). were randomly selected and sub-cultured. In addition,
Out of the poplar clones 741, Brauna11, T89, W52 and endophytic isolates were obtained from the transgenic
Esch5, as well as the larch- and spruce clones, five lines of the poplar clone 741, clone 73 and the two
individual plants were used for the analysis of black locust clones by placing plant segments on R2A
endophytic bacteria. medium.
Plant material of 250 mg was ground in 1 ml of The phylogenetic analysis of endophytic isolates
sterile 0.85% NaCl with a sterile mortar and pestle. was based on 16S rDNA sequencing and performed as

Table 1 Culturable endophytic bacteria in in vitro plant tissue cultures of woody plants
Plant clonea Origin of plant tissue culture Population density (cfu/g FW)b Taxonomic assignment of isolatesc

Poplar
741 Y.C. Tian (Beijing) 9.8 9 105 Paenibacillus (8)
d 4
Brauna11 M. Fladung (GHD) 2.3 9 10 Paenibacillus (8)
T89 M. Fladung (GHD) 9.5 9 104 Paenibacillus (8)
W52 M. Fladung (GHD) 1.2 9 107 Paenibacillus (8)
Esch5 M. Fladung (GHD) 3.0 9 106 Paenibacillus (8)
73 M. Yang (Baoding) Not tested Paenibacillus (1), Agrobacterium rubi (1)
741/2 (cryI + API) Y.C. Tian (Beijing) Not tested Methylobacterium (2)
741/3 (cryIII) Y.C. Tian (Beijing) Not tested Methylobacterium (2), Paenibacillus (4)
741/4 (cryI) Y.C. Tian (Beijing) Not tested Paenibacillus (5)
741/5 (cryIII) Y.C. Tian (Beijing) Not tested Bacillus (1), Paenibacillus (4)
741/6 (cryI + API) Y.C. Tian (Beijing) Not tested Bacillus (3), Paenibacillus (1)
741/7 (cryI + API) Y.C. Tian (Beijing) Not tested Bacillus (2), Paenibacillus (3)
741/12 (cryI + API) Y.C. Tian (Beijing) Not tested Bacillus (1), Stenotrophomonas (2)
741/15 (cryI) Y.C. Tian (Beijing) Not tested Paenibacillus (2)
Larch
HL 86/6 D. Ewald (WSD)d 2.1 9 103 Paenibacillus (8)
SELA 30/4 D. Ewald (WSD) 2.0 9 106 Paenibacillus (8)
6
LSP2 D. Ewald (WSD) 1.4 9 10 Paenibacillus (8)
Spruce
Marienberg D. Ewald (WSD) 1.1 9 103 Paenibacillus (8)
Black locust
Rob2498 I. Zaspel (WSD) Not tested Paenibacillus (2)
Rob2402 I. Zaspel (WSD) Not tested Paenibacillus (1)
a
741 $ [Populus alba L. 9 (P. davidiana Dode + P. simonii Carr.) 9 P. tomentosa Carr.], Esch5 $ (P. tremula L. 9 P.
tremuloides Michx.), Brauna11 $ (P. tremula L.), T89 # (P. tremula L. 9 P. tremuloides Michx.), W52 # (P. tremula L.), 73
[(Populus tomentosa Carr. 9 Populus bolleana Louche) 9 Populus tomentosa Carr.] (Yang et al. 2006), eight lines obtained from
poplar clone 741 carrying a modified cryI or cryIII gene and the arrowhead proteinase inhibitor (API) gene (Tian et al. 2000)
b
Mean values from five individual plants were calculated
c
The assignment of the isolates was based on the BLAST analysis and on the Classifier programme of the RDP. The numbers of
isolates for the identified genera or species are given in brackets
d
GHD—Großhansdorf; WSD—Waldsieversdorf

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described by Ulrich et al. (2008). For the taxonomic In all clones tested, high amounts of endophytic
placement of endophytic isolates, single strand bacteria were found with clones W52 and Esch5
sequencing of about 400–600 bp was applied. Repre- showing the highest densities (Table 1). All isolates
sentative Paenibacillus isolates were chosen for from five out of the six poplar clones, the larch, spruce
sequencing both complementary strands of nearly and black locust clones were found to belong to the
full-length 16S rDNA (1,500 bp). genus Paenibacillus (Table 1). Only in clone 73 and in
To test the effect of Paenibacillus on in vitro plant six of the transgenic lines of clone 741, endophytic
growth explants of the poplar clone 741 free from isolates of other genera could be found. Altogether,
culturable bacteria were used. These explants were Paenibacilli represented 59% of the endophytic bac-
obtained from regenerated shoot meristems from teria in these lines. Representative Paenibacillus
glasshouse grown plants. Microcuttings were planted isolates of all poplar clones, as well as larch, spruce
on the in vitro medium BEMB200 (Ewald and and black locust, were chosen for a detailed phyloge-
Süss 1993) in 100 ml Erlenmeyer flasks after expos- netic analysis. Four clusters could be identified within
ing the basal 5 mm stem part to an overnight TSA the genus as shown in the phylogenetic tree (Fig. 1).
broth culture of the Paenibacillus isolate 22 (final The majority of isolates showed a strong similarity to
concentration 2 9 107 cfu ml-1) for 2 s. The control P. humicus (99.5%). The second group including the
cuttings were given a quick dip in TSA. Twenty-five isolates obtained from black locust, indicated a close
bottles, each with one plant per treatment were tested. relationship to P. pabuli. Isolates from the transgenic
Root length, number of main roots and sprout height poplar line 741/15 clustered with P. polymixa and P.
were recorded 2 and 3 weeks after inoculation. The peoriae. The isolate P117 from poplar clone 73
colonisation of P. humicus in the plants was deter- showed the highest similarity to P. motobuensis. Five
mined by grinding roots and shoots of five of the poplar clones, the larch clones and the spruce
representative plants in 0.85% NaCl and plating on clone were only occupied by bacteria belonging to the
R2A. The experiment was repeated twice with the P. humicus-group.
same number of replications.

Fig. 1 Phylogenetic tree

showing the relationship
of the 16S rRNA gene
sequences of representative
endophytic Paenibacillus
isolates obtained from
in vitro cultures of different
woody plants. The tree was
generated using the
Neighbor-Joining algorithm
using Bacillus subtilis
(DQ529249) as the
outgroup. Type strains
of Paenibacillus species
were used for the
calculations. The origin of
the isolates is given in
brackets. The 16S rRNA
gene sequences obtained in
this study were deposited in
the EMBL nucleotide
sequence database under the
Accession Nos.
AM906085–AM906090.
Bar, 0,01 relative sequence
divergences

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Table 2 Effect of the inoculation of microcuttings of poplar clone 741 with Paenibacillus strain P22 on in vitro root development at
3 weeks after inoculation
P. humicus strain 22 Control Significance

Number of roots 3.68 1.96 *

Average root length (mm) 147.64 89.68 **
Sprout height (mm) 42.20 38.76 Not significant
Data were subjected using the Proc genmod (SAS 9.1.3.) programme
*Significant at P \ 0.05(v2-test); ** significant at P \ 0.05 (t-test)

Compared to the endophytic bacterial community vitreous. Endophytic colonization with the P. humicus
of field grown poplars (Ulrich et al. 2008; Moore isolate 22 was comparable for the shoots and roots
et al. 2006), the diversity found in the micropropa- 3 weeks after inoculation with 2.1 9 106 and
gated cultures was very low and the composition 1.0 9 106 cfu, respectively.
completely different. Leifert et al. (1989) reported In conclusion, this study revealed the importance
that in newly introduced cultures diverse Gram- of the Paenibacillus strains as endophytic bacteria in
negative bacteria were found, whereas in older micropropagated tissue cultures of woody plants. The
micropropagated material 70% of Gram-positive results indicated the potential of the Paenibacillus
bacteria could be detected. About 90% of the older strains as plant growth promoting endophytic bacteria
cultures contained only one bacterial species, indi- under in vitro conditions for poplar, but further
cating that the special in vitro conditions may result research is needed for investigating the mechanisms
in the excessive growth of certain populations of of growth promotion and potential effects on ex vitro
endophytic bacteria or single strains. These results do plants.
correspond with the high abundance of Paenibacillus,
especially P. humicus, in this study. All clones tested Acknowledgements This work was supported by grant
0313285I from the Federal Ministry of Education and
have been micropropagated for at least 5 years.
Research. We are grateful to Mrs. H. Enkisch for her
Paenibacillus, which has also been detected as an excellent technical assistance. We also thank Prof. Yang
endophytic bacterium in field-grown poplar clones from the Agricultural University Hebei, Baoding for providing
and therefore may be introduced via plant cuttings the poplar clone 741 including several transgenic lines and the
white poplar clone, and Dr. M. Fladung and Dr. I. Zaspel for
seems to accumulate well under the special in vitro
providing plant materials from poplar and black locust clones,
conditions. respectively.
Paenibacillus has been found as an endophyte in
different woody plants like pine, coffee and poplar
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