RESEARCH ARTICLE

E¡ects of plant genotype and growth stage on the structure

of bacterial communities associated with potato
(Solanum tuberosum L.)
Leo van Overbeek1 & Jan Dirk van Elsas2
1
Plant Research International, Wageningen, The Netherlands; and 2Department of Microbial Ecology, Centre for Ecological and Evolutionary Studies,
Groningen University, Haren, The Netherlands

Correspondence: Leo van Overbeek, Plant Abstract

Research International, Droevendaalsesteeg
1, 6708PB Wageningen, The Netherlands.
Tel.: 131 317 476041; fax: 131 317 418094;
e-mail: leo.vanoverbeek@wur.nl
Received 21 December 2007; revised 28
January 2008; accepted 28 January 2008.
First published online 18 March 2008.
DOI:10.1111/j.1574-6941.2008.00469.x
Editor: Jim Prosser
Keywords
genetically modified plants; rhizosphere;
endophytes; bacterial communities; cultivar;
plant growth stage.
The effects of genotype, plant growth and experimental factors (soil and year) on
potato-associated bacterial communities were studied. Cultivars Achirana Inta,
Désirée, Merkur and transgenic Désirée line DL12 (containing T4 lysozyme gene)
were assessed in two field experiments. Cross-comparisons between both experi-
ments were made using Désirée plants. Culture-dependent and -independent
approaches were used to demonstrate effects on total bacterial, actinobacterial and
Pseudomonas communities in bulk and rhizosphere soils and endospheres. PCR-
denaturing gradient gel electrophoresis fingerprints prepared with group-specific
primers were analyzed using multivariate analyses and revealed that bacterial
communities in Achirana Inta plants differed most from those of Désirée and
Merkur. No significant effects were found between Désirée and DL12 lines. Plant
growth stage strongly affected different plant-associated communities in both
experiments. To investigate the effect of plant-associated communities on plant
health, 800 isolates from rhizospheres and endospheres at the flowering stage were
tested for suppression of Ralstonia solanacearum biovar 2 and/or Rhizoctonia solani
AG3. A group of isolates closely resembling Lysobacter sp. dominated in young
plants. Its prevalence was affected by plant growth stage and experiment rather
than by plant genotype. It was concluded that plant growth stage overwhelmed any
effect of plant genotype on the bacterial communities associated with potato.

Seghers et al., 2004), soil history (Smalla et al., 2001; Gu &

Introduction
The preservation of beneficial plant-associated microbial
communities to support plant health is an important issue
in agriculture, especially in the light of the need to enhance
sustainability. Agricultural management measures can in-
duce clear shifts in the structures of plant-associated micro-
bial communities (Garbeva et al., 2004). For example, plant
genotype (cultivar) can exert strong effects on the bacterial
communities that associate with plants (Germida &
Siciliano, 2001; Adams & Kloepper, 2002; Gu & Mazzola,
2003). Thus, differences in the densities and structures of
endophytic microbial communities have been observed in
different cultivars of cotton (Adams & Kloepper, 2002).
Other factors believed to play important roles as effectors
of plant-associated microbial communities are land use
(Peters et al., 2003; Garbeva et al., 2004; Salles et al., 2004;
Mazzola, 2003; Garbeva et al., 2006) and plant growth stage
(Duineveld et al., 1998). It is actually expected that the
combined action of plant and environmental factors will
determine the structure of plant-associated microbial com-
munities. To allow any agricultural exploration of these
communities, for instance in respect of an enhancement of
antagonism against plant pathogens, it is of utmost rele-
vance to pinpoint the key factors that influence their
structures. On the basis of a sound selection of plant cultivar
as well as agricultural practices, new strategies may thus be
developed to control plant pests and diseases (Berg et al.,
2002).
The introduction of transgenic crops equipped with genes
that control bacterial or fungal pathogens, such as potato
lines with T4 lysozyme biosynthetic genes (Porsch et al.,
1998; Lottmann et al., 1999; Smalla et al., 2001; Heuer et al.,

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2002), may develop into a major strategy to combat plant
diseases. However, crops that express such rather broad-
spectrum disease-suppressive traits have the potential to
cause undesirable effects on nontarget and potentially
beneficial microbial communities. In particular, adverse
effects on microbial communities that are involved in
natural pathogen suppression might affect plant health
(Lottmann et al., 1999; Heuer et al., 2002). However,
field experiments that were recently performed in Spain
with the novel T4-lysozyme producing potato line
DL12, which was also used in this study, provided a first
indication that the rhizosphere microbial communities,
as far as the experiment could discern, were hardly
affected by the presence of the transgene (Rasche et al.,
2006b). Instead, it was found that field location and
plant growth stage exerted much stronger effects than
T4-lysozyme.
The bacterial groups that are known to contribute to
plant health, including disease suppression and plant health
support, are taxonomically diverse, as are the mechanisms
involved. Members of species belonging to the genera
Actinomyces (Zinniel et al., 2002; Castillo et al., 2003;
Coombs et al., 2004), Bacillus (Benhamou et al., 1998; Bacon
& Hinton, 2002; Reva et al., 2002), Burkholderia (Balandreau
et al., 2001; Salles et al., 2004), Lysobacter (Islam et al., 2005),
Pseudomonas (Duijff et al., 1997; Gu & Mazzola, 2003;
Rediers et al., 2003) and Serratia (Press et al., 1997;
Benhamou et al., 2000; Tan et al., 2001; Berg et al., 2002;
Kamensky et al., 2003) have been found to play major roles
in plant health support. The mechanisms by which
these bacteria influence plant health may involve direct
antibiosis of pathogens (Thomashow & Weller, 1988;
Mavrodi et al., 2006), competition for nutrients with patho-
gens (Deacon, 1991), induction of systemic resistance in
plants (Van Loon et al., 1998) or any possible combination
of these mechanisms.
Although it is likely that as-yet-uncultured populations
also play a role in plant health support, so far there is little
knowledge about them. Thus, modern studies on beneficial
plant-associated microbial communities should also place a
special focus on the nonculturable organisms. Current
molecular fingerprinting techniques such as PCR-denatur-
ing gradient gel electrophoresis (DGGE) are well suited to
study plant-associated communities including those resid-
ing at the plant–soil interface (Duineveld et al., 1998) and
inside plants (Garbeva et al., 2001; Seghers et al., 2004).
Thus, specific primers can be used to assess the roles of
groups of organisms in disease suppression, e.g. members
of the taxa Actinobacteria (Heuer et al., 1997; Sessitsch et al.,
2002; Postma et al., 2005; Hunter et al., 2006), Pseudomonas
(Thomashow & Weller, 1998; Reiter et al., 2003; Garbeva
et al., 2004), Bacillus (Garbeva et al., 2003) and Burkholderia
(Salles et al., 2002). Such assessments have the potential to
Journal compilation

c 2008 Federation of European Microbiological Societies
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L. van Overbeek & J.D. van Elsas
provide a greater insight into the fluctuations of these
populations in their association with plants under selected
agricultural regimes.
The aim of this study was to assess the effects of plant
genotype and growth stage on potato-associated bacterial
communities in the field. The relevance of these effects for
populations that is potentially responsible for plant health
support are assessed. Therefore, isolates were tested for
their capacities to suppress the potato pathogens Ralstonia
solanacearum biovar (bv) 2 and Rhizoctonia solani AG3.
A novel T4 lysozyme-producing potato line, DL12 (Porsch
et al., 1998), was included in this study because of its
presumed effect on plant-associated bacterial communities.
The effects of three potato cultivars, Désirée, Merkur and
Achirana Inta, were analyzed in a first field experiment and
those of two selected potato lines, Désirée and the transgenic
line DL12, in a second one. The effects of location and
year were then analyzed using Désirée plants from both
experiments.
Materials and methods
Potato plants and field experiments
In vitro ‘explants’ (plantlets grown from surface-sterilized
stem nodes) of potato (Solanum tuberosum L.) cultivar
Achirana Inta (A) were provided by Dr Angela Sessitsch,
ARC Seibersdorf, Austria, whereas those of cultivars Désirée
(D) and Merkur (M) were obtained from commercial
stocks. In vitro explants of potato line DL12, cultivar D
(which expresses a bacteriophage T4 lysozyme gene con-
trolled by the CaMV 35S promoter), were kindly provided
by Dr Andreas Mahn, MPB Cologne, Germany. Minitubers
of cultivars A, D and M, as well as of line DL12, propagated
from in vitro explants and grown in potting soil in the
greenhouse, were kindly provided by Dr Jan van der Haar,
HZPC Research, Metslawier, the Netherlands. Before plant-
ing in the field, the minitubers were stored at 12 1C for 3
months, in order to break dormancy. Two field experiments,
both with the same experimental design albeit with different
potato cultivars and lines, were performed as described
below.
Experiment 1
To compare the effect of cultivar on plant-associated bacter-
ial communities, minitubers of potato cultivars A, D and
M were planted in an agricultural field (the previous crop
was pasture) at a location near Emmeloord, Northeast
polder, the Netherlands (field 1) in May 2002. The soil of
field 1 was characterized as a sandy clay loam (clay content,
35%; organic matter content, 2.2%; pH-KCl, 7.3; and water-
holding capacity, 38%).
FEMS Microbiol Ecol 64 (2008) 283–296
Effects on bacterial communities associated with potato
Experiment 2
To compare the effect of two selected potato lines, including
the one equipped with the T4 lysozyme gene, on the plant-
associated microbial communities, minitubers of lines D
and DL12 were planted in an agricultural field (previous
crop was pasture) in Marknesse, the north-east polder, the
Netherlands (field 2) in May 2003. The soil of field 2 was also
characterized as a sandy clay loam (clay content, 32%;
organic matter content, 2.5%; pH-KCl, 7.5; and water-
holding capacity, 36%). Cross-comparisons between experi-
ments 1 and 2 were made between D plants grown in both
fields.
Experimental design and sampling
Planting of the minitubers of each cultivar was in experi-
mental plots (2 m  2 m) into soil ridges of about 25 cm in
height. Four replicate plots per cultivar or line were
randomly distributed over total areas of, respectively,
120 m2 (‘Experiment 1’) and 96 m2 (‘Experiment 2’). The
experimental fields were enclosed by borders of 2 m width.
Soil treatments, planting and crop treatments (fungicide
treatments and water application during dry periods) were
carried out in accordance with potato production schemes
common in the Netherlands. During crop growth, plants
were regularly monitored for growth and the occurrence of
stress, pests and diseases. At the flowering stage (stage 6
according to the potato development systematic of Hack
et al., 1993), flowers were removed from D and DL12 plants
grown in ‘Experiment 2’ for biosafety reasons.
Samples were obtained from roots with adhering soil
(rhizosphere) and stems (endosphere) of individual plants.
One representative plant per experimental plot was sampled
at young, flowering and senescing growth stages, respec-
tively, 1, 6 and 9 (Hack et al., 1993). Bulk soil samples (12 g)
were obtained from the top soil (5–20 cm), between the
ridges. One sample was obtained per experimental plot, at
times coinciding with plant growth stages 1, 6 and 9.
Sample treatment and processing
Bulk soil samples were split into two portions. One portion
(0.5 g) was frozen and stored at  70 1C for later DNA
extraction. The other portion (10 g) was suspended in 95 mL
of sterile 0.1% sodium pyrophosphate (NaPP) solution in
250-mL Erlenmeyer flasks with 10 g gravel (2–4 mm dia-
meter) and shaken for 10 min at 200 r.p.m. at room
temperature. To obtain rhizosphere soil and endosphere
samples, plants were treated as follows: roots with adhering
soil from each plant were split into two portions:
one portion (about 1 g) was directly frozen and stored at
 70 1C and the other one (about 3 g) was ground and
shaken in 95 mL of sterile 0.1% NaPP solution in 250-mL
FEMS Microbiol Ecol 64 (2008) 283–296
285
Erlenmeyer flasks with 10 g gravel (10 min, 200 r.p.m., room
temperature). Plant stem parts were cut off at 1–5 cm above
the soil level. These parts (about 3 g plant1) were washed
and surface-sterilized, first by rinsing in running tap water
to remove adhering soil, then by immersion in 1.5% NaClO
solution for 1 min, followed by immersion in 96% ethanol
for 1 min and a final wash in sterile tap water for 1 min. The
surface-sterilized stem parts were aseptically peeled by
removal of the epidermal layer with a sterile scalpel. One
part of the remaining tissue (about 1 g) was stored frozen at
 70 1C, whereas the other part (about 2 g) was transferred
to a sterile plastic bag containing 5 mL sterile 0.1% NaPP.
The latter sample was crushed by smashing with a hammer,
after which the resulting homogenates were shaken with
3 mL sterilized water.
Plate counts and dual-culture assays
The suspensions made from bulk soil and rhizosphere and
crushed stems were serially (10-fold) diluted in 0.1% NaPP
and plated onto R2A plates (BD, Franklin lakes, NJ). Plates
were incubated for 5 days at 27 1C, after which colonies were
counted. Twenty-five isolates per rhizosphere soil sample
and 15 per stem sample, of five cultivars over both fields
with four replicates per cultivar and resulting in a total of
800 isolates, were selected from plates of the highest
dilutions and streaked to purity on R2A.
Dual-culture tests for antagonism towards R. solanacear-
um bv 2, strain1609, were performed on plates with strain
1609 cells embedded in R2A agar. Therefore, molten R2A
agar kept at 45 1C was mixed with exponentially grown
and washed strain 1609 cells at a density of about Log
8 cells mL1, and plates were allowed to cool down. Cells
from isolates were then streaked onto the agar surface, after
which plates were incubated for 10 days at 27 1C to allow
colony growth. Antagonists were identified by clearance
zones of between 1 and 3 mm in diameter that appeared
around colonies. For testing of antagonism to R. solani AG3,
cells from single isolates were aseptically transferred to R2A
plates, after which an agar plug with R. solani AG3 mycelium
was placed at 2 cm distance from each streak. Plates were
incubated for 10 days and antagonists were identified by
inhibition of R. solani AG3 mycelial growth in zones around
the colonies, varying between 1 and 15 mm in diameter.
Each isolate was tested twice in the antagonism assays for
both pathogens. Isolates that showed reproducible antagon-
ism to either or both pathogens were scored positively for
antagonism. Antagonists were maintained in 20% glycerol
at  70 1C.
Identification of antagonists
Genomic DNA was extracted from all antagonists using the
genomic extraction procedure described in Ausubel et al.
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286
(1998). PCR amplifications were performed on 10 ng of
genomic DNA in standard 50-mL reaction mixtures accord-
ing to Van Elsas & Wolters (1995), using primers 968F and
1401R (Heuer et al., 1997; Heuer & Smalla, 1999). PCR
amplification was performed in a PTC-100 (MJ Research
Inc., MA) thermocycler. PCR products with the expected
length (about 450 bp) were cloned into the pGEM-T vector
(Promega, Leiden, the Netherlands) and introduced into
Escherichia coli JM109 by transformation, according to the
protocol provided by the manufacturer. Clones with the
expected 450-bp inserts were subjected to sequencing with
universal M13 primers using the service of Greenomics,
Plant Research International B.V., the Netherlands. DNA
sequences were checked for chimeras using the Check-
Chimera tool, after which the nonchimeric sequences were
assessed for similarities to database sequences available at
the NCBI web site (http://www.ncbi.nlm.nih.gov) using the
basic local alignment sequence tool (BLAST). DNA sequences
of isolate Lysobacter sp. HV177 were deposited in the EMBL
database (http://www.ebi.ac.uk) under accession number
AM849108.
DNA extractions from bulk soil, rhizosphere and
endosphere
Total bulk and rhizosphere soil DNA was extracted from the
frozen samples using the MoBio UltraClean soil DNA
isolation kit (MoBio Laboratories, BIOzym TC, Landgraaf,
the Netherlands). Total endosphere DNA was extracted from
frozen surface-sterilized stem parts according to Garbeva
et al. (2001), with the inclusion of a preceding homogeniza-
tion step by crushing (with a hammer) of the stems in 3 mL
0.1% NaPP in sterilized plastic bags. DNA extracts from all
samples were stored in TE buffer (Tris-HCl, 10 mM; EDTA,
1 mM; pH 8) and kept at  20 1C.
PCR-DGGE analyses
PCR was performed on 2–10 ng total community DNA
(bulk and rhizosphere soil) and 5–20 ng of stem part DNA.
Primers 968F, with a GC clamp (Muyzer et al., 1993), and
1401R were used to amplify a 450-bp fragment of bacterial
16S rRNA genes. PCR for bacteria was performed as in
Van Elsas & Wolters (1995). Furthermore, semi-nested PCR
systems were used to amplify partial 16S rRNA genes from
Pseudomonas and Actinobacteria spp., respectively. For Pseu-
domonas spp., the semi-nested system of Garbeva et al.
(2004) (involving primers PsR, PsF and 968F with GC
clamp) was used to amplify a roughly 300-bp fragment of
Pseudomonas 16S rRNA genes. For Actinobacteria, the semi-
nested system of Heuer et al. (1997) consisting of primers
243F, 1378 and 984F with a GC clamp, which amplifies a
400-bp fragment of Actinobacteria spp. 16S rRNA genes, was
used. All PCR products were diluted in TE buffer to a final
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c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Dutch government works
L. van Overbeek & J.D. van Elsas
concentration of 20 ng mL1. The DGGE procedure was
performed according to Garbeva et al. (2006). For separa-
tion of total bacterial and Pseudomonas bands, a gradient of
45–65% and for actinobacterial bands of 30–60% was used.
Migration distance and intensity of individual bands in
the fingerprints were chosen as the parameters used for
calculation of the Shannon diversity index (H 0 ). The Shan-
non diversity index combines richness and evenness of
bacterial species and increases with the number of species
(individual bands in fingerprints) and the more even
distribution of biomass (total band size) over the different
bacterial species in each sample. The same parameters were
also chosen for multivariate analyses. On one occasion, an
individual band was sliced from DGGE gel, PCR amplified
with bacterial primers and cloned into pGEM-T. Following
an identity check on DGGE, the resulting clone inserts
were sequenced and analyzed for similarities to database
sequences available at the NCBI web site by BLAST searching.
Statistical analyses
Statistical analyses were performed on the log CFU numbers
and Shannon diversity (H 0 ) values from bulk and rhizo-
sphere soil and endosphere samples using ANOVA. Four
replicates per treatment were used and the effects of treat-
ments (using genotype and growth stage) on log CFU
numbers or diversity values were considered to be distin-
guishable at a confidence level of 95%. For bulk soil log CFU
numbers, comparisons were made between growth stages 1,
6 and 9 separately for each experiment. For rhizosphere
and endosphere log CFU numbers, comparisons were made
between growth stages 1, 6 and 9 of A, D and M plants
(‘Experiment 1’) and of D and DL12 plants (‘Experiment 2’)
and between corresponding samples of D plants taken at
the three growth stages from both experiments. The diver-
sity values calculated from PCR-DGGE fingerprints were
compared for each primer system (bacteria, Pseudomonas or
Actinobacteria) separately. For bulk soil, comparisons were
made between samples taken at the three growth stages per
experiment. For the rhizosphere and endosphere, four
comparisons were made: (1) between growth stages 1, 6
and 9 of D plants from experiment 1, (2) between A, D and
M plants at growth stage 6 from ‘Experiment 1’, (3) between
D and DL12 plants at growth stages 1, 6 and 9 from
‘Experiment 2’ and (4) between corresponding samples of
D plants at growth stages 1, 6 and 9 from both experiments.
Multivariate analyses (Ter Braak, 1987) on the same PCR-
DGGE fingerprints were applied by making use of CANOCO
software (CANOCO 4.5; Biometris, Wageningen, the Neth-
erlands). The same comparisons made to determine effects
in species diversity were also applied in multivariate ana-
lyses. Comparisons were only made between fingerprints
produced with the same primer system. The positions and
FEMS Microbiol Ecol 64 (2008) 283–296
Effects on bacterial communities associated with potato 287

Table 1. Log CFU numbers and number of antagonists in potato rhizospheres and endospheres of two experiments
Log CFU g1 dry soil or g plant (number of antagonists/total number of isolates tested)

Experiment 1 Experiment 2

Plant growth stage at sampling Achirana Inta Désirée Merkur Désirée DL12
Rhizosphere
Growth stage 1 (young) 6.87A 7.40B 7.57B 8.89H 9.01H
Growth stage 6 (flowering) 7.97A (20/100) 8.59C (44/100) 8.42C (2/100) 9.22I (70/100) 9.14I (51/100)
Growth stage 9 (senescent) 8.30C 8.63C 8.44C 9.11I 9.10I
Endosphere
Growth stage 1 (young) 4.79O 3.75N 5.48O 2.90X 2.93X
Growth stage 6 (flowering) 6.16O (1/60) 6.69P (10/60) 6.14O (1/60) 4.00X (36/60) 3.33X (34/60)
Growth stage 9 (senescent) 6.53P 7.24P 7.03P 6.27Y 5.95Y
Growth stages according to potato plant development systematic of Hack et al. (1993). Statistical differences (P o 0.05) are indicated by different
letters: rhizospheres experiment 1, A–C; rhizospheres experiment 2, H, I; endospheres experiment 1, O, P; endospheres experiment 2, X, Y.

intensities of the bands in individual fingerprints were
considered to be ‘species’, and the growth stage and cultivar
‘environmental’ factors. Detrended correspondence analysis
(DCA) was used to calculate the lengths of the ordination
axes of the species response curves. A calculated length of the
ordination axis between 0 and 4 SDs indicates a linear
relationship and would justify the use of redundancy
analysis (RDA). The existence of no relationship between
species and environmental factors was considered to be the
null hypothesis. A Monte-Carlo permutation test based on
499 random permutations was included to establish statis-
tical significance. The effects of the environmental factors on
the PCR-DGGE fingerprints were considered to be distin-
guishable at a 95% or a higher confidence level.
Results
Plant development
Experiment 1
Potato cultivars A, D and M, grown in field 1, showed slight
differences in development. Plant growth stage 1 occurred
between 21 and 25 days for D and M, and between 23 and 26
days for A. Furthermore, growth stage 6 occurred between
60 and 65 days for D, between 62 and 66 days for M and
between 70 and 73 days for A. Finally, growth stage 9 was
between 130 and 140 days for D, between 135 and 145 days
for M and between 150 and 160 days for A. Hence, in field 1,
A plants were between 2 and 20 days later in flowering and
tuber setting than those of D, whereas M developed almost
synchronously with D.
Experiment 2
Potato lines D and DL12, grown in field 2, were in growth
stage 1 between 20 and 25 days, in growth stage 6 between
50 and 55 days and in growth stage 9 between 140 and 150
days. This was roughly similar for both D and DL12 plants
and thus we concluded that D and DL12 plants developed
synchronously.
CFU numbers in bulk and rhizosphere soils and
endospheres
Experiment 1
In bulk soil, the log CFU numbers for bacteria were between
7.51 and 7.64 g1 dry soil and there were no significant
differences between the three growth stages. Log CFU
numbers in the rhizosphere soils increased progressively
with plant developmental stage: from 6.87 to 8.30 for A,
from 7.57 to 8.44 for D and from 7.40 to 8.63 for M plants
(Table 1). Log CFU numbers in A rhizospheres were
significantly lower than in those of D or M at growth stages
1 and 6. The log CFU numbers in the D and M rhizospheres
did not differ from each other at all three growth stages, but
log CFU numbers in both rhizospheres were significantly
higher at growth stages 6 and 9 than at growth stage 1. In the
endosphere, log CFU numbers increased progressively with
plant growth stage, that is, from 4.79 to 6.53 for A, from 3.75
to 7.24 for D and from 5.48 to 7.03 for M plants (Table 1). At
growth stage 1, the log CFU numbers in the endospheres
were significantly lower in D than in A and M plants. At
growth stage 6, however, these numbers were significantly
higher in D than in A and M plants, whereas at growth stage
9, log CFU numbers in the three cultivars did not differ from
each other.
Experiment 2
In bulk soil, the log bacterial CFU numbers ranged from
7.95 to 8.18 g1 of dry soil. There were no significant
differences in these numbers between the three growth

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288 L. van Overbeek & J.D. van Elsas

stages. In the rhizosphere, the log CFU numbers increased

during plant development, i.e. from 8.89 to 9.11 for D and
from 9.01 to 9.10 for DL12 plants between, respectively,
growth stages 1 and 9 (Table 1). The log CFU numbers in the
D and DL12 rhizospheres at growth stages 1, 6 and 9 did not
differ from each other (per time point), whereas log CFU
numbers at growth stage 1 were significantly lower than
those at growth stages 6 and 9. This indicates that there were
no differences between the log CFU numbers in the rhizo-
spheres of both lines, but there was bacterial growth over the
course of plant development, which was the same for D and
DL12. The log CFU numbers in the endospheres also
increased progressively during plant development. From M B1 B1 B6 B6 B9 B9 1 1 1 1 6 6 6 6 9 9 9 9 M
growth stages 1 to 9, these numbers increased, respectively,
from 2.90 to 6.27 for D and from 2.93 to 5.95 for DL12 Fig. 1. Bacterial PCR-DGGE fingerprints of bulk soils at growth stage 1,
plants. The endospheric log CFU numbers were not sig- 6 and 9 (respectively, B1, B6 and B9 in figure) and rhizosphere of Désirée
from field 1 at growth stages 1, 6 and 9 (respectively, 1, 6 and 9 in
nificantly different between the two plant lines. However,
figure). The marker (M), consists of 16S rRNA gene amplicons from
they differed over time, as at growth stages 1 and 6 the Enterobacter cloaceae BE1, Listeria innocua ALM 105, Arthrobacter sp.
numbers were significantly lower than those at growth AR1 and Burkholderia cepacia P2. The gel was loaded with two of four
stage 9. replicate bulk soil samples and all four replicate rhizosphere samples to
assess variation and allow within-gel comparisons.

Molecular microbial community fingerprint

analyses
Table 2. Phylotype diversity (H 0 ) of PCR-DGGE profiles made from
rhizosphere and endosphere samples of two field experiments
Experiment 1
Phylotype diversity (H 0 )
0
The phylotype diversities (H ) in the bulk soil, as calculated
Rhizosphere Endosphere
from the PCR-DGGE fingerprints, varied between 2.03 and
2.96 for total bacteria, between 1.18 and 2.46 for Pseudomo- Bac Psew Act Bac Psew Actw
nas and between 0.96 and 3.04 for Actinobacteria. Although Experiment 1
variation in phylotype diversity occurred at different plant Désirée at all growth stages
growth stages, there were no significant differences between Growth stage 1 2.58 1.82B 1.86 1.82 2.53 1.18
the fingerprints from bulk soils for total bacterial (Fig. 1) Growth stage 6 2.67 1.53AB 1.93 1.53 2.60 1.46
Growth stage 9 2.67 0.99A 1.50 0.99 2.61 1.19
and Pseudomonas and Actinobacteria. The phylotype diver-
All cultivars at growth stage 6
sities in the rhizosphere varied between 2.58 and 2.82 for Achirana Inta 2.58 1.99 2.34 2.69 2.17B 2.43
total bacteria, between 0.99 and 2.09 for Pseudomonas and Désirée 2.82 2.09 2.92 2.56 1.75A 2.99
between 1.50 and 2.92 for Actinobacteria (Table 2). There Merkur 2.73 1.85 2.56 2.71 2.00AB 3.16
was no significant effect of cultivar type on the diversity of Experiment 2
the different bacterial groups in the rhizosphere. However, Désirée (D) and DL12 at all growth stages
a significant effect of plant growth stage was found: the D, growth stage 1 2.81 2.81 2.07 2.65 2.53B 1.81B
D, growth stage 6 2.91 2.91 1.99 2.62 2.60B 1.53B
diversity in the Pseudomonas fingerprints of D plants at
D, growth stage 9 2.74 2.84 2.10 2.55 2.10A 0.99A
growth stage 1 was higher than those at growth stages 6 and DL12, growth stage 1 2.86 2.90 2.08 2.70 2.55B 1.86B
9. This indicates that the diversity of Pseudomonas species DL12, growth stage 6 2.84 2.96 2.09 2.64 2.61B 1.66B
decreased over the time of plant growth. DL12, growth stage 9 2.99 2.86 2.06 2.57 2.05A 0.86A
Multivariate analyses of the PCR-DGGE fingerprints Primers systems used: Bac, total bacteria; Pse, Pseudomonas; Act,
from bulk soils confirmed the observations made with
Actinobacteria. wSignificant difference (P o 0.05) where A o B.
phylotype diversity, namely that no significant effects of
plant growth stage were found with the three applied primer
systems. Comparisons of bulk and rhizosphere soils of D plants were significantly different from those in rhizospheres
plants at corresponding growth stages revealed significant of D and M plants (Table 3). This indicates a cultivar effect
differences in total bacterial, Pseudomonas and actinobacter- on these communities in the rhizosphere. Furthermore,
ial communities between these two compartments. Bacterial at growth stages 6 and 9, the respective communities of D
and actinobacterial communities in the rhizospheres of A plants were significantly different from those at growth stage

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Effects on bacterial communities associated with potato 289

Table 3. Effect of plant growth stage and plant genotype on bacterial community structures in potato rhizospheres and endospheres of two field
experiments
Variable with P-value

Parameters PCR primer system Rhizosphere Endosphere

Experiment 1
Growth stage (only Désirée) Total bacterial Growth stage 1 0.004 Growth stage 1 0.05
Growth stage 6 0.05 Growth stage 6 4 0.05
Growth stage 9 4 0.05 Growth stage 9 4 0.05
Pseudomonas Growth stage 1 0.02 Growth stage 1 4 0.05
Growth stage 6 4 0.05 Growth stage 6 4 0.05
Growth stage 9 4 0.05 Growth stage 9 4 0.05
Actinobacteria Growth stage 1 0.004 Growth stage 1 4 0.05
Growth stage 6 0.002 Growth stage 6 4 0.05
Growth stage 9 4 0.05 Growth stage 9 4 0.05
Cultivar (only growth stage 6) Total bacterial Achirana Inta 0.03 Achirana Inta 0.02
Désirée 4 0.05 Désirée 4 0.05
Merkur 4 0.05 Merkur 4 0.05
Pseudomonas Achirana Inta 4 0.05 Achirana Inta 0.004
Désirée 4 0.05 Désirée 4 0.05
Merkur 4 0.05 Merkur 4 0.05
Actinobacteria Achirana Inta 0.03 Achirana Inta 4 0.05
Désirée 4 0.05 Désirée 4 0.05
Merkur 4 0.05 Merkur 4 0.05
Experiment 2
Growth stage and cultivar Total bacterial Growth stage 1 0.002 Growth stage 1 4 0.05
Growth stage 6 0.002 Growth stage 6 4 0.05
Growth stage 9 4 0.05 Growth stage 9 4 0.05
Désirée 4 0.05 Désirée 4 0.05
DL12 4 0.05 DL12 4 0.05
Pseudomonas Growth stage 1 4 0.05 Growth stage 1 0.002
Growth stage 6 4 0.05 Growth stage 6 4 0.05
Growth stage 9 0.01 Growth stage 9 4 0.05
Désirée 4 0.05 Désirée 4 0.05
DL12 4 0.05 DL12 4 0.05
Actinobacteria Growth stage 1 0.05 Growth stage 1 4 0.05
Growth stage 6 4 0.05 Growth stage 6 4 0.05
Growth stage 9 4 0.05 Growth stage 9 0.05
Désirée 4 0.05 Désirée 4 0.05
DL12 4 0.05 DL12 4 0.05

1. Also, the bacterial and actinobacterial communities at
growth stage 9 in D plants were significantly different from
those at growth stage 6. Thus, there was a strong effect of
growth stage on the bacterial communities in D rhizosphere
soils.
The phylotype diversity in the endosphere, as derived
from the PCR-DGGE fingerprints, varied between 0.99 and
2.71 for total bacteria, between 1.75 and 2.61 for Pseudomo-
nas and between 1.18 and 3.16 for Actinobacteria. The
diversity in the Pseudomonas community of A plants was
higher than in those of D and M plants, although significant
differences were only present between A and D (Table 2).
Multivariate analyses of the endosphere PCR-DGGE finger-
prints revealed that the bacterial and Pseudomonas commu-
nities in the A plants were significantly different from those
in D and M plants (Table 3). Hence, there was a clear effect
of cultivar on the potato endosphere communities. The
bacterial communities in the endosphere of D plants at
growth stages 6 and 9 were significantly different from those
at growth stage 1 (Table 3); hence, growth stage exerted a
small effect on the bacterial communities in the endosphere
of D plants.
Experiment 2
The phylotype diversity (H 0 ) in bulk soil derived from the
respective PCR-DGGE fingerprints was between 2.58 and
3.14 for total bacteria, between 1.93 and 2.00 for Pseudomo-
nas and between 2.48 and 3.04 for Actinobacteria. There
were no significant differences in diversity between the

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290 L. van Overbeek & J.D. van Elsas

different plant growth stages in any of the three groups
assessed. The diversity inferred from the rhizosphere soil
fingerprints of both plant lines was between 2.74 and 2.99
for total bacteria, between 2.81 and 2.96 for Pseudomonas
and between 1.99 and 2.10 for Actinobacteria (Table 2). For
none of the three bacterial groups significant differences in
phylotype diversity were found in the D and DL12 plant
rhizospheres at growth stages 1, 6 and 9.
Multivariate analyses performed on the PCR-DGGE
fingerprints from bulk soils (three primer systems) con-
firmed the absence of differences between the fingerprints at
three growth stages for the two plant lines tested. However,
the fingerprints of bulk and rhizosphere soils of D plants
at corresponding growth stages revealed significant differ-
ences for all three bacterial groups assessed. There were no
significant differences between both plant lines in the
fingerprints made with the three primer systems at three
growth stages (Table 3). Only a small and ephemeral line-
specific effect (P = 0.12) was found between the actinobac-
terial fingerprints of both lines at growth stage 6, which was
not apparent anymore at growth stage 9. Significant differ-
ences between the growth stages of both lines were observed
in fingerprints at growth stage 1 vs. 6 and 9 for bacteria and
Actinobacteria, between growth stages 6 and 9 for bacteria
and between 1 and 6 vs. 9 for Pseudomonas. This indicates
that a clear plant line-specific effect on the bacterial com-
munities in the potato rhizospheres was absent, whereas
there was a strong effect of plant growth stage on the
different rhizosphere communities of both lines.
In the endosphere of both potato lines, the phylotype
diversity varied between 2.55 and 2.70 for total bacteria,
between 2.05 and 2.61 for Pseudomonas and between 0.86
and 1.86 for Actinobacteria (Table 2). Per time point, no
significant differences in the diversity of these three groups
were found between lines D and DL12. The phylotype
diversity decreased with the growth stage, and the Pseudo-
monas and actinobacterial diversities were significantly low-
er at growth stage 9 than at growth stages 1 and 6.
Multivariate analyses of the PCR-DGGE fingerprints, per
bacterial group assessed at the three growth stages, revealed
the absence of significant differences between lines D and
DL12 (Table 3). However, for both lines significant effects
of growth stage were found in the Pseudomonas and
actinobacterial fingerprints (Table 3). There was, thus, no
plant line-specific effect on the different bacterial commu-
nities in the potato endospheres, but there was a clear effect
of plant growth stage on endosphere communities of both
lines.
Cross-comparison between D plants from both
experiments
For all three bacterial groups analyzed, the multivariate
analyses of the PCR-DGGE fingerprints of D plant rhizo-
spheres (three growth stages) revealed significant differences
in particular between the two experiments. Moreover,
comparisons between the respective endosphere fingerprints
also revealed significant effects of experiment (data not
shown). This indicates that experiment (and the dependent
factors field and year) exerted strong effects on the three
bacterial communities assessed in the rhizospheres and
endospheres of D plants.
Identification of a specific bacterial population
In ‘Experiment 2’, a conspicuous dominating band was
observed in the bacterial PCR-DGGE fingerprints from all

* * * * * * * *
L M D1 D1 D1 T1 T1 T1 D6 D6 D6 T6 T6 T6 D9 D9 D9 T9 T9 T9 M L

Fig. 2. Bacterial PCR-DGGE fingerprints of Désirée (D) and DL12 (T) rhizospheres of plants from field 2 at growth stages 1, 6 and 9 (respectively, 1, 6
and 9 in figure). The same marker (M) in was used as in Fig. 1 and a 16S rRNA gene amplicon of isolate HV177 (identified as Lysobacter sp.) was included
(L). Arrows indicate the band comigrating with that of isolate HV177. This band was present in lanes marked with (). The gel was loaded with three (out
of four) replicate samples per growth stage to obtain best resolution. A band comigrating with that of HV177 was found in the fourth samples of
Désirée and DL12 rhizospheres at growth stage 1 and not in those at growth stages 6 and 9.

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Effects on bacterial communities associated with potato
replicate rhizosphere samples of the D and DL12 lines at
growth stage 1 (Fig. 2). This band was absent from the
corresponding bulk soil fingerprints. Moreover, it was
incidentally present in the rhizospheres and endospheres of
D and DL12 plants at growth stages 6 and 9. Independent
repetition of PCR and DGGE with the same samples
revealed identical results, indicating that the presence of
these bands did not result from any experimental artifact. A
comparison of the DGGE fingerprints with clamped PCR
products prepared from key dominating bacterial isolates
(see later) revealed that the position of the conspicuous
band coincided with that of the Lysobacter sp. isolate HV177
16S rRNA gene-derived amplicon. Thus, evidence was
obtained for the selection of a Lysobacter HV177-affiliated
population at different growth stages in the D and DL12
rhizospheres. Sequencing of clones prepared from excised
band material from the growth-stage-1 D rhizosphere
fingerprints indeed revealed the prevalence among the
clones of sequences showing 4 99% identity with Lysobac-
ter sp. isolate HV177.
At growth stage 1, the relative population sizes of this
presumed Lysobacter sp. in the rhizospheres of both lines
(inferred from the relative band intensities) were between
5% and 7% of the total band intensity in all three D plants
and between 8% and 13% in all three DL12 plants (Fig. 2).
Moreover, it was 13% in one D rhizosphere at growth stage 6
and 10% in one DL12 rhizosphere at growth stage 9. In the
endosphere, the relative population size was 15% in one D
plant at growth stage 6 and 20% in one DL12 plant at
growth stage 9. This band was not found in any of the
cultivars grown in ‘Experiment 1’, either in the rhizosphere
or in the endosphere. The appearance of a large population
of organisms affiliated with Lysobacter sp. HV177 was
therefore associated with the potato plants growing in
‘Experiment 2’, and might thus be field-specific.
Screening of isolates for antagonism against
potato pathogens
Incidence of antagonists
Inhibition of growth of R. solani AG3 and R. solanacearum
bv 2 strain 1609 was observed in a total of 269 out of 800
colonies selected from rhizospheres and endospheres of
growth-stage-6 plants of both experiments. Of the 269
antagonists, 187 originated from rhizospheres (500 isolates
screened) and 82 from the endosphere (300 screened). The
relative abundance of antagonists was, thus, slightly higher
in the rhizosphere than in the endosphere. In the rhizo-
sphere, the highest antagonist incidences were found in D
plants from ‘Experiment 2’ and the lowest in M plants; in
the endosphere, the highest incidences were in D plants
from ‘Experiment 2’ and the lowest in A and M plants (Table
FEMS Microbiol Ecol 64 (2008) 283–296
291
1). Overall, the antagonist incidences in ‘Experiment 1’
(three cultivars) were lower than those in ‘Experiment 2’
(two lines).
Among the antagonists, those suppressing R. solani AG3
were most frequently found (219), followed by R. solana-
cearum bv 2 suppressors (26) and organisms that suppressed
both pathogens (24) (Table 4). Antagonists against R. solani
AG3 were found in the rhizospheres of all cultivars and lines
in both experiments (152) and in the endospheres of both
lines in ‘Experiment 2’ (67). Antagonists against R. solana-
cearum were found in the rhizospheres of cultivars D and M
(experiment 1) and in both lines from ‘Experiment 2’, as well
as in the endospheres of all cultivars from ‘Experiment 1’
and in that of D from ‘Experiment 2’. Isolates suppressing
both pathogens were only found in D and DL12 plants, i.e.
in the rhizospheres of D (both experiments) and DL12 and
in the endosphere of DL12.
Identification of isolates
Analysis of the 16S rRNA gene sequences of the 269
antagonistic isolates showed that 241 strains resembled
defined cultured species at or above the threshold level for
species identity of 97% similarity. The remaining 28 se-
quences showed hits at levels between 86% and 96% with
database entries and were thus considered to be remotely
related to defined species in the database. All 269 isolates
belonged to only four phyla, i.e. the Proteobacteria (13
genera, 164 antagonists), Actinobacteria (five genera, 70
antagonists), Firmicutes (three genera, 26 antagonists) and
Bacteroidetes (one genus, nine antagonists) (Table 4). The
241 well-identifiable antagonists were distributed among 45
different lower (genus)-level taxonomic groups. Eight of
these groups encompassed at least six isolates, as follows in
order of decreasing frequency of isolation: Lysobacter sp.
(76), Pseudomonas fluorescens (21), Bacillus mycoides (16),
Streptomyces caviscabies (13), Lentzea flavoverrucosus (12),
Serratia plymuthica (10), Serratia lateritius (10) and Flavo-
bacterium columnaris (6). In this context, no prevalence was
found for antagonist types to cultivars or lines, in both
experiments. Antagonists belonging to the Lysobacter sp.
cluster occurred in the rhizospheres of A, D (both experi-
ments) and DL12, as well as in the endosphere of A, M and
DL12 plants. We singled out one dominating isolate,
HV177, as the model strain to analyze PCR-DGGE patterns
for its prevalence (see before).
Discussion
Genotype (cultivar and line), plant growth stage and experi-
ment (combined effect of field and year, only assessed for
cultivar D) were found to be the three factors affecting
the composition of the bacterial communities associated
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292 L. van Overbeek & J.D. van Elsas

Table 4. Antagonists against Rhizoctonia solani AG3 and/or Ralstonia solanacearum bv 2 from rhizospheres and endospheres of different plant
genotypes
Number of antagonists

Rhizosphere Endosphere

Experiment 1 Experiment 2 Experiment 1 Experiment 2

Nearest match Achirana Inta Désirée Merkur Désirée DL12 Achirana Inta Désirée Merkur Désirée DL12
Proteobacteria
Pedobacter sp. 1(f)
Delftia sp. 1(f), 2(bf) 1(bf)
Herbaspirillum sp. 2(f)
Janthinobacterium sp. 1(f)
Buttiauxella sp. 1(b)
Erwinia sp 3(b) 1(b)
Kluyvera sp 2(b) 2(b)
Pantoea sp. 1(f)
Lysobacter sp. 3(f) 14(f) 24(f) 28(f) 1(b) 1(b) 5(f)
Pseudomonas sp. 1(f) 2(b), 8(f) 1(f) 12(f) 2(f) 2(b) 7(f) 10(f)
Rahnella sp. 2(b)
Serratia sp. 1(f) 2(b), 3(f) 2(f),1(b) 9(f) 2(f)
Stenotrophomonas sp. 2(f) 1(f)
Actinobacteria
Arthrobacter sp. 1(b) 1(b)
Kitasatosporia sp. 1(b)
Lentzea sp. 11(f) 1(f)
Micromonospora sp. 1(f)
Streptomyces sp. 3(f) 5(f), 4(bf) 8(f), 10(bf) 7(f), 5(bf) 11(f) 1(bf)
Firmicutes
Bacillus sp. 1(f) 2(f) 7(f) 12(f)
Paenibacillus sp. 1(bf)
Lactococcus sp. 3(b)
Bacteriodetes
Flavobacterium sp. 5(f) 2(f) 2(f)
Nearest match (97% similarity or higher) with 16S rRNA gene sequences in public database (http://www.ncbi.nlm.nih.gov). Abbreviations for
pathogens: b, R. solanacearum bv 2 antagonist; f, R. solani AG3 antagonist; bf, antagonistic against both pathogens.

with potato. Cultivation-dependent and -independent ap-
proaches were applied to assess the most important factors
affecting the bacterial communities associated with the
potato plants. Both methods showed the same result,
namely that plant growth stage and experiment exerted the
strongest effects on the potato-associated bacterial commu-
nities. Potato genotype also contributed to the plant-asso-
ciated community structures, but a clear effect was only
observed between cultivars A vs. D and M and no effects
were found between D and DL12 lines. These data primarily
indicate that the plant-associated bacterial communities
change over time during plant development and that
this dynamic behavior occurred in all cultivars tested. The
bacterial communities in the D plants differed from each
other between the two experiments and this is most prob-
ably related to the fact that the composition of plant-
associated bacterial communities is mainly determined by
the soil (Garbeva et al., 2004) and that the soil communities
in both fields were different. Communities associated with
plants that are growing in different fields possibly never
converge to a structure that is ‘common’ or prototypical for
any cultivar in particular, and hence those from the same
cultivar are likely to be different in different soils.
Next to total bacterial communities, two other commu-
nities were investigated in particular because of their pro-
posed contributions to plant health, namely Pseudomonas
and Actinobacteria (Thomashow & Weller, 1988; Coombs
et al., 2004; Garbeva et al., 2004; Postma et al., 2005; Hunter
et al., 2006). Perhaps, not surprisingly, these communities
were affected by cultivar, plant growth stage and experiment
at about the same level as the total bacterial communities.
Moreover, the appearance of a bacterial population closely
related to isolate HV177 – which suppressed both R. solani
AG3 and R. solanacearum bv 2 and was identified as a
Lysobacter sp. – was also influenced by these factors. Isolates
affiliated with HV177 belonged to the largest group of
antagonists found in this study. They closely resembled
antagonists from other studies classified as Lysobacter

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Effects on bacterial communities associated with potato
enzymogenes (Folman et al., 2003) and Lysobacter sp. strain
SB-K88 (Islam et al., 2005). Bacterial types that potentially
support plant health were thus affected in the same way as
other bacterial populations. The variation in the number of
Lysobacter-type isolates between individual plants high-
lighted their heterogeneous distribution over different
plants in the fields. Presumably, the colonization and estab-
lishment of this group of organisms in potato plants are
stochastic processes that depend to a large extent on the
chances of successful interactions between individual Lyso-
bacter sp. cells and emerging potato roots. As a consequence,
the colonization process may appear to be erratic, and only
those plants that were colonized by members of this group
might be well protected against invading pathogens. Other
antagonistic groups of microorganisms, possibly including
fungal species found in the same potato cultivar (Götz et al.,
2006), may also contribute to plant protection and it is very
likely that these populations are distributed in the same way
as the Lysobacter sp. types over individual plants.
Differences in bacterial endophytic and other plant-
associated communities between plant species or between
cultivars of the same species have been observed before
(Latour et al., 1996; Germida & Siciliano, 2001; Adams &
Kloepper, 2002; Gu & Mazzola, 2003). We anticipated the
potentially greatest effects of plant genotype, in particular
on the endophytic bacterial communities, in the T4 lyso-
zyme-producing line DL12. This line was actually selected
because of its effect on the pathogen Erwinia carotovora
subspecies atroseptica, which was stronger than that in other,
comparator, transgenic lines (Rasche et al., 2006a, c). It was
shown that the T4 lysozyme not only exerts bactericidal
activity by cell wall degradation but also broader micro-
bicidal activity against a wide spectrum of fungal and
bacterial pathogens (Düring et al., 1999). Expression of the
T4 lysozyme gene in root tissue of potato line DL12 was
shown to occur by reverse transcriptase-PCR analysis of
lysozyme-specific mRNA (F. Rasche, pers. commun.). To
our surprise, a large effect of DL12 plants on endosphere or
rhizosphere bacterial communities could not be found,
except for a small and ephemeral effect on the actinobacter-
ial community in the rhizosphere.
Our data confirmed the conclusions made before in
studies in which a preceding generation of T4 lysozyme-
producing potato lines was analyzed, namely that at most
slight and temporal effects on the bacterial communities
associated with potato are to be expected (Heuer & Smalla,
1999; Lottmann et al., 1999, 2001; Heuer et al., 2002). The
general lack of pronounced effects of T4 lysozyme in potato
on endophytic bacterial communities was also observed by
Rasche et al. (2006c). The absence of a clear difference in the
prevalence of particular bacterial groups in lines DL12 vs. D
indicates that, within the confines of the detection metho-
dology, the presence of the T4 lysozyme biosynthetic gene in
FEMS Microbiol Ecol 64 (2008) 283–296
293
DL12 does not affect any specific bacterial target at signifi-
cant levels. The reason for this lack of effect might lie in a
supposed ‘inaccessibility’ of the released T4 lysozyme for
local bacterial populations, or in an accelerated degradation
of the lysozyme by proteases that are locally present.
Another possibility is that only a small fraction of the
microbial populations associated with genetically altered
crops is influenced by the T4 lysozyme, whereas factors like
plant growth stage and environment affect a larger part of
the local community and thus overwhelm this effect. This
may be the reason why, in general, it is so difficult to detect-
effects of plant genotype on the composition of plant-
associated communities, especially when dealing with trans-
genic plants (Schmalenberger & Tebbe, 2002; Milling et al.,
2004; O’Callagan et al., 2004).
The results obtained in this study are in line with those
obtained in a field experiment performed in Spain in which
the same transgenic line, DL12, was used (Rasche et al.,
2006b). In this study, it was shown that the effects caused by
field location and growth stage on shoot-associated bacterial
communities were stronger than that of the transgenic line.
Also, in a field trial conducted with other transgenic plants,
Griffiths et al. (2006) showed that soil type and plant growth
stage exerted the strongest effects on soil microbial commu-
nities. They also assessed target and nontarget micro-
arthropods associated with a transgenic maize line equipped
with the cry1Ab (Bt) gene. Generalizing this issue, it
becomes clear that, in many cases, plant growth and soil
factors exert stronger effects on the microbial communities
associated with plants than does plant genotype. In our
study, we demonstrated that these factors exerted effects on
the bacterial communities that are present in the rhizo-
sphere as well as inside plants, which opens a new perspec-
tive on the factors driving endophyte diversity.
In conclusion, plant growth stage and experiment
(field and year), which were the factors that contributed
most to the variations in the potato-associated bacterial
communities in most, but not all cases, overwhelmed
the variation caused by differences in plant genotype. On
the other hand, genotype-specific variations in the
plant-associated communities were found, and these might
conceptually be linked to differences in root exudate pat-
terns (amount and type of compounds). In this context,
selection of specific plant genotypes to improve healthy
plant development might still be a viable option. In parti-
cular, the apparent selection of antagonists like Lysobacter
sp. types in two potato lines, even at a small window
of opportunity, might offer possibilities for advances in
potato phytopathogen control. Plant health can thus be
further improved by measures that selectively favour
the colonization and establishment of beneficial popula-
tions. Appropriate cultivar selection can be helpful in this
process.
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294
Acknowledgements
This work was supported by the EU Fifth framework
program ‘Quality of life and management of living re-
sources’ under program QLK3-CT-2000-01598, acronym
‘POTATOCONTROL’, and the Dutch Ministry of Agricul-
ture, Nature and Food Quality DWK 352 program ‘Agro-
biodiversity’. We thank Henk Velvis for excellent technical
support, Dr Jan van der Haar, HZPC Research, Metslawier,
for providing potato minitubers, Dr Andreas Mahn, MPB
Cologne for providing transgenic potato line DL12, Dr
Angela Sessitsch, ARC Seibersdorf Research GmbH, Austria,
for providing Achirana Inta plants and Dr Joanna Salles for
helpful discussions.
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