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In vitro antagonists of Rhizoctonia solani tested on lettuce:

rhizosphere competence, biocontrol e⁄ciency and rhizosphere
microbial community response
Modupe F. Adesina1, Rita Grosch2, Antje Lembke1, Tzenko D. Vatchev3 & Kornelia Smalla1
Julius Kühn-Institut – Federal Research Centre for Cultivated Plants (JKI), Braunschweig, Germany; 2Institute for Vegetables and Ornamental Crops
(IGZ), Großbeeren, Germany; and 3Department of Plant Pathology and Immunology, Plant Protection Institute, Sofia, Bulgaria

Correspondence: Kornelia Smalla, Julius Abstract

Kühn-Institut – Federal Research Centre for
Cultivated Plants (JKI), Messeweg 11/12,
The rhizosphere competence of 15 in vitro antagonists of Rhizoctonia solani was
D-38104 Braunschweig, Germany. Tel.: 149 determined 4 weeks after sowing inoculated lettuce seeds into nonsterile soil.
531 299 3814; fax: 149 531 299 3006; Based on the colonization ability determined by selective plating, eight strains were
e-mail: selected for growth chamber experiments to determine their efficacy in controlling
bottom rot caused by R. solani on lettuce. Although in the first experiment all
Received 30 July 2008; revised 18 March 2009; antagonists colonized the rhizosphere of lettuce with CFU counts above 2  106 g 1
accepted 24 March 2009. of root fresh weight, only four isolates significantly reduced disease severity. In
Final version published online June 2009.
subsequent experiments involving these four antagonists, only Pseudomonas
jessenii RU47 showed effective and consistent disease suppression. Plate counts
and denaturing gradient gel electrophoresis (DGGE) fingerprints of Pseudomonas-
Editor: Jim Prosser
specific gacA genes amplified from total community DNA confirmed that RU47
established as the dominant Pseudomonas population in the rhizosphere of
Keywords inoculated lettuce plants. Furthermore, the DGGE fingerprint revealed that
Rhizoctonia solani; antagonists; lettuce; R. solani AG1-IB inoculation severely affected the bacterial and fungal community
rhizosphere competence; biocontrol; microbial structure in the rhizosphere of lettuce and that these effects were much less
communities. pronounced in the presence of RU47. Although the exact mechanism of antag-
onistic activity and the ecology of RU47 remain to be further explored, our results
suggest that RU47 is a promising agent to control bottom rot of lettuce.

stitute or a supplement to reduce the use of chemical

Introduction pesticides (Sweetingham, 1996; Compant et al., 2005). Over
Rhizoctonia solani Kühn, the anamorph of Thanatephorus the last decades, bacteria with the ability to suppress
cucumeris (Frank) Donk, is a widespread soil-borne fungus R. solani have been isolated from different soils (Weller
comprising plant parasitic or saprophytic strains (Ogoshi, et al., 2002; Faltin et al., 2004; Garbeva et al., 2004; Berg
1987; González Garcı́a et al., 2006). The species affects many et al., 2005; Adesina et al., 2007). However, many studies
important agricultural and horticultural crops worldwide, have reported variabilities in the performance of biological
causing diseases such as black scurf, crown rot and bottom control agents (BCA) and lack of a correlation between in
rot (Ogoshi, 1996). The control of the pathogen is difficult vitro inhibition tests and the field performance of BCA
because of its wide host range and its ability to survive as (Milus & Rothrock, 1997; Schottel et al., 2001). It is assumed
sclerotia under adverse environmental conditions. In prac- that the major contributors to this inconsistency in planta
tice, the control of diseases caused by R. solani relies mainly are the inherent characteristics of the BCA such as poor root
on fungicides (Kataria & Gisi, 1996). However, increasing colonization or insufficient production of antifungal meta-
concern about the health and environmental hazards asso- bolites at the pathogen infection sites due to variable
ciated with the use of agrochemicals has resulted in the expression of genes involved in disease suppression (Raaij-
search for viable alternatives. Hence, biological control makers & Weller, 2001; Haas & Défago, 2005; Raaijmakers
involving the use of microorganisms that can suppress or et al., 2008). Recently, the application of molecular
antagonize plant pathogens is being considered as a sub- DNA-based methods contributed toward an improved

c 2009 Federation of European Microbiological Societies FEMS Microbiol Ecol 69 (2009) 62–74
Published by Blackwell Publishing Ltd. All rights reserved
Biocontrol of Rhizoctonia solani and rhizosphere competence 63

understanding of the ecology of BCA in the rhizosphere (75 mg mL 1). The 15 rifampicin-resistant mutant strains were
where the BCA interact not only with the plant and the tested for inhibitory activity against two highly pathogenic
pathogen but also with the indigenous microbial commu- isolates belonging to the R. solani anastomosis groups AG1-IB
nity (Götz et al., 2006; Scherwinski et al., 2008). and AG2 (Faltin et al., 2004; Grosch et al., 2004). In vitro
In this study, 15 strains were selected from a collection of inhibitory activity against R. solani AG1-IB and AG2 was
bacterial isolates with in vitro antagonistic activity towards determined essentially as described by Adesina et al. (2007).
R. solani AG3 (the causal agent of black scurf on potato) and/ The mutant strains were stored at 80 1C in Luria–
or Fusarium oxysporum (which causes wilting disease in Bertani broth (ROTH, Germany) containing 20% glycerol
diverse crops), which were previously isolated and character- supplemented with rifampicin (75 mg mL 1).
ized (Adesina et al., 2007). The in vitro antagonists that
originated from four disease-suppressive soils located in Lettuce rhizosphere colonization assays
France, the Netherlands, Sweden and the United Kingdom
To determine the rhizosphere competence of the 15 rifam-
were first screened for their ability to colonize the rhizosphere
picin-resistant strains, rhizosphere colonization assays were
of lettuce in nonsterile soil. Based on their colonization ability,
performed in nonsterile soil under greenhouse conditions.
eight strains were selected for growth chamber experiments.
Lettuce seeds (cultivar ‘Tizian’, Syngenta, Bad Salzuflen,
The work presented here aimed to investigate the potential of
Germany) were surface sterilized in 2% sodium hypochlor-
the in vitro antagonists to suppress bottom rot disease caused
ite (NaOCl) for 5 min, followed by three subsequent wash-
by R. solani AG1-IB on lettuce plants in vivo (growth chamber
ing steps with sterile distilled water. The seeds were
experiments) and to study the survival and root colonization
pregerminated on sterile moistened filter papers in sterile
efficiency of the antagonists by means of selective plating. The
Petri dishes for 2 days at room temperature. Pregerminated
abundance of the most promising antagonist and the response
lettuce seeds were soaked in a cell suspension (cell concen-
of the indigenous fungal and bacterial communities in the
tration adjusted to c. 109 cells mL 1) of each bacterial
rhizosphere to plant inoculation were evaluated by cultiva-
antagonist for 1 h. Four pregerminated and inoculated seeds
tion-independent molecular fingerprints. This is one of the
per pot were sown into nonsterile potting soil [turf sub-
few studies assessing not only biocontrol efficiency but also
strate/clay granulate No. 4230, Klasmann-Deilmann GmbH,
the survival of the bacterial inoculants and effects on the
Geeste, Germany, sieved (2-mm mesh width) and mixed
indigenous microbial communities.
with 80% (w/w) sand]. The pots were kept in the greenhouse
at 20 1C and 30% humidity. Each treatment was replicated
Materials and methods three times. Four weeks after sowing, the plants were care-
fully removed from the soil and vigorously shaken. The
Bacterial and fungal strains roots with adhering soil were placed in a Stomacher plastic
Fifteen out of 248 antagonists isolated from four suppressive bag and processed after adding sterile saline mechanically by
soils (Adesina et al., 2007) were selected on the basis of Stomacher blending for 3 min at high speed. CFU of the
either their (1) strong in vitro activity against R. solani AG3 inoculant per gram of root fresh weight (rfw) were deter-
(isolate Ben3; host plant, potato) or (2) activity towards mined by plating serial dilutions of the rhizosphere suspen-
both R. solani AG3 and F. oxysporum (isolate Foln3; host sions on R2A supplemented with rifampicin (75 mg mL 1)
plant, flax). All bacterial antagonists used in this study were and cycloheximide (100 mg mL 1). The CFU were counted
maintained on R2A agar (Difco, Detroit, MI). Rhizoctonia after 2 days of incubation at 28 1C.
solani AG1-IB (isolate 7/3; host plant, lettuce) and AG2
(isolate W4; host plant cabbage) were obtained from the Growth chamber experiments
strain collection of the Institute for Vegetables and Orna-
Based on the results of the rhizosphere colonization assays,
mental Crops, Großbeeren, Germany) and maintained on
eight antagonistic isolates with the ability to colonize lettuce
Waksman agar containing 5 g of proteose-peptone (Merck,
roots at cell densities of approximately log CFU 4 4 g 1 of
Darmstadt, Germany), 10 g of glucose (Merck), 3 g of meat
rfw or more were selected for the growth chamber experi-
extract (Chemex, München, Germany), 5 g of NaCl (Merck),
ments. The efficacy of eight antagonistic isolates in control-
20 g of agar (Difco), and distilled water (to 1 L) (pH 6.8).
ling bottom rot disease caused by R. solani AG1-IB on
lettuce plants (cultivar ‘Tizian’) was assessed under favor-
Generation of antibiotic-resistant mutants
able conditions for the pathogen in the growth chamber
To facilitate reisolation of the bacterial inoculants from the (York, Mannheim, Germany; 16 h/8 h day/night cycle,
rhizosphere, spontaneous rifampicin-resistant mutants of the 500 mmol m 2 s 1, 20/15 1C and 60%/80% relative humid-
bacterial antagonists were generated by plating overnight ity). In experiment 1, all eight in vitro antagonists were
cultures onto R2A agar supplemented with rifampicin evaluated. Four isolates with the best disease suppression

FEMS Microbiol Ecol 69 (2009) 62–74

c2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
64 M.F. Adesina et al.

were included in experiments 2 and 3, while only two For each treatment and sampling time three symptomless
isolates with the best results in experiments 1 and 2 were plants were used. Loosely adhering soil was removed from
assessed in experiment 4. In experiment 3, the effect on plant the roots by shaking the plants. Five grams of roots with
growth was determined based on lettuce shoot and root dry adhering soil were suspended in 20 mL of sterile saline and
weight, in treatments without pathogen inoculation. shaken vigorously in sterile flask containing six glass beads
Seeds were treated with bacterial antagonists directly before (0.6 mm in diameter) on a rotary shaker for 1 h at 307 r.p.m.
sowing. Each antagonist was grown overnight on nutrient agar Aliquots of the rhizosphere suspension were immediately
supplemented with rifampicin at 75 mg mL 1. Overnight- processed for enumeration of the inoculated strains. Serially
grown colonies were resuspended in a sterile 0.3% NaCl diluted rhizosphere suspensions were plated on R2A med-
solution and shaken to obtain a homogeneous cell suspension. ium (Difco) supplemented with rifampicin (75 mg mL 1)
The concentration was adjusted in a spectrophotometer to a and cycloheximide (100 mg mL 1). The remaining rhizo-
density corresponding to c. 109 CFU mL 1. Lettuce seeds were sphere suspension was centrifuged at 13 000 g for 5 min.
soaked in the bacterial suspension for 1 h at room temperature. After discarding the supernatants, the cell pellets were stored
Seeds that were incubated in sterile saline for the same time frozen at 20 1C.
served as a control. Seeds were germinated in a seedling tray The ability of the most promising antagonist RU47 to
containing 92 holes filled with a nonsterile mixture of quartz colonize the vascular tissue of lettuce roots and leaves was
sand and substrate [Fruhsdorfer Einheitserde Typ P, Germany; determined at 7 weeks after sowing in experiment 3. Roots
chemical analysis (mg per 100 g): N = 75, P = 75, K = 125; from the inoculated plants were shaken to remove loosely
pH 5.9] at a 1 : 1 ratio (v/v). Lettuce plantlets were transferred adhering soils and washed in running water. One gram of
at the two- to three-leaf stage to pots filled with the same soil the washed roots or leaves were soaked in 1% sodium
mixture with one plant per pot. Drenching of lettuce plants hypochlorite for 1 min. Surface-sterilized root or leaf sam-
with bacterial antagonists was carried out 24 h after transplant- ples were rinsed four times with sterile distilled water and
ing. Each plant of the treatment inoculated with RU47 and macerated using a sterile mortal and pestle. The suspensions
R. solani (RU471Rs) was drenched with 20 mL of the bacterial of the macerated roots or leaves were serially diluted and
suspension (107 CFU mL 1) and the noninoculated healthy plated as mentioned above.
control (Ctrl) and inoculated with R. solani (Ctrl1Rs) treat-
ments received 20 mL of sterile saline. The pathogen was added DNA extraction from rhizosphere samples
on barley kernels infested with the R. solani AG1-IB (isolate 7/3;
Total community (TC)-DNA was obtained from the rhizo-
Schneider et al., 1997) 1 day after drenching the plants with
sphere pellets by means of the Bio101 extraction kit
bacterial antagonists. Five kernels per lettuce seedling were
(Q.BIOgene, Carlsbad, CA) after a harsh lysis step. The TC-
placed 1 cm deep at a distance of 2 cm from the lettuce plant.
DNA was purified using the GENECLEAN Spin kit (Q.BIO-
Noninfested barley kernels were added to the control treat-
gene). DNA yields were estimated after electrophoresis in
ments accordingly. The pots were watered daily to maintain the
1% agarose gel stained with ethidium bromide under UV
substrate moisture. Each treatment consisted of six replicates
light in comparison with the 1-kb gene-rulerTM DNA
(four pots per replicate) arranged in a randomized design. Nine
ladder (Fermentas, St Leon-Rot, Germany) run on the same
additional pots (three plants per time point and treatment)
agarose gel. Depending on the yield of the extracted TC-
were included for microbial analysis.
DNA, dilutions were made with sterile milliQ water between
Suppression of bottom rot disease by bacterial 1 : 50 and 1 : 100 (c. 1–5 ng) and were used as a template for
inoculants PCR amplification of bacterial 16S rRNA gene fragments.
Undiluted TC-DNA, or 1 : 10 dilutions (c. 20 ng DNA)
Seven weeks after sowing (4 weeks after pathogen inocula-
served as a template for PCR of fungal 18S rRNA gene
tion), the experiments were terminated and a total of 24
plants were evaluated for suppression of bottom rot disease
of R. solani AG1-IB based on (1) the disease incidence and
PCR amplification of 16S and 18S rRNA and gacA
on (2) the effect on the shoot dry weight (SDW). The disease
gene fragments for denaturing gradient gel
incidence was assessed by recording the number of plants
electrophoresis (DGGE) analysis
with visual symptoms or the number of dead plants. The
shoot dry weight of each lettuce plant was measured. To amplify the 16S rRNA gene fragments of Pseudomonas
from TC-DNA the nested PCR system described by Milling
Root colonization by bacterial inoculants
et al. (2004) was used. Diluted amplicons obtained from the
To determine the survival and root colonization efficiency first PCR served as templates for a second PCR using the
of the inoculated antagonists, rhizosphere samples were bacterial primers F984-GC/R1378 and PCR conditions
collected at three time points: 3, 5 and 7 weeks after sowing. described by Heuer et al. (1997). To generate bacterial

c 2009 Federation of European Microbiological Societies FEMS Microbiol Ecol 69 (2009) 62–74
Published by Blackwell Publishing Ltd. All rights reserved
Biocontrol of Rhizoctonia solani and rhizosphere competence 65

community fingerprints, the latter primer system and Results

conditions were used to directly amplify 16S rRNA gene
fragments from TC-DNA. Amplification of the Pseudomonas- In vitro activity of antagonists and colonization
specific gacA gene fragment (575 bp) was performed of lettuce root
using the nested PCR approach described by Costa et al.
To facilitate monitoring of the 15 in vitro antagonistic
(2007). Amplification of 18S rRNA gene fragments
strains by selective plating, rifampicin-resistant mutants
(1650 bp) was done using the primer pair NS0/EF3
were generated for all antagonists. The ability of the 15
(Messner & Prillinger, 1995; Smit et al., 1999) in a first PCR
in vitro antagonists to colonize the roots of lettuce plants
assay, followed by a second PCR step with the primer pair
was determined 4 weeks after sowing inoculated pregermi-
NS1/FR1GC (White et al., 1990; Vainio & Hantula, 2000).
nated lettuce seeds into nonsterile soil. Eight strains with
The PCR conditions used were described by Costa et al.
CFU counts 4 4  103 g 1 rfw were selected for subsequent
growth chamber experiments. The in vitro antagonistic
activities of these eight strains towards representatives of
the R. solani anastomosis groups AG1-IB (host plant,
DGGE lettuce), AG2 (host plant, sugar beet) and AG3 (host plant:
DGGE analysis was performed using the Dcode System potato) are given in Table 1. Five antagonists (KS16, KS90,
apparatus (Bio-Rad Inc., Hercules, CA). The polyacrylamide KS70, KS74 and KF36) showed strong in vitro inhibitory
gel was a gradient gel containing 9% (fungi) or a double activity against R. solani AG1-IB (the model pathogen used
gradient gel of 6–9% (bacteria, Pseudomonas and gacA) in this study for the growth chamber experiments) with
acrylamide with a denaturant gradient of 18–38% inhibition zones larger than 6 mm while strain RN86
(fungi) or 26–58% (bacteria, Pseudomonas and gacA) of expressed a moderate inhibitory activity of o 6 mm zone
denaturants according to Gomes et al. (2005) (where 100% of inhibition. Only Serratia marcescens AFNS31 and Pseudo-
denaturants contain 7 M urea and 40% formamide). Ali- monas jessenii RU47 displayed weak activity against R. solani
quots of PCR samples (2–4 mL) were applied to the DGGE AG1-IB with inhibition zones o 3 mm after 5 days of
gels, and the run was performed in 1  Tris-acetate-EDTA incubation. Information on the identity of the strains
buffer at 58 1C with a constant voltage of 220 V for 6 h selected for the growth chamber experiment, their in vitro
(bacteria, Pseudomonas, gacA) or 180 V for 18 h (fungi). The antagonistic activity and their secondary metabolite pro-
DGGE gels were silver stained, according to Heuer et al. duction is given in Table 1.

Survival of the antagonists in the rhizosphere

Cluster analysis of DGGE fingerprints of lettuce plants under growth chamber
Fungal, bacterial, Pseudomonas and gacA-based Pseudomonas conditions
DGGE community fingerprints were analyzed using the Although similar cell densities of bacterial suspension were
software package GELCOMPAR II version 5.6 (Applied Maths, used for seed inoculation (c. 109 mL 1), the cell densities of
Kortrijk, Belgium). Background was subtracted using a antagonists on the seed surfaces showed remarkable differ-
rolling disk method with an intensity of 10 (relative units), ences and were in the range of 8  104– 8  107 CFU per seed
and the lanes were normalized. (data not shown). However, the bacterial inoculants actively
A dendrogram was constructed by the Pearson correlation colonized the lettuce roots. The CFU counts of the inoculant
index for each pair of lanes within a gel and cluster analysis strains determined 3 weeks after sowing ranged between
by the unweighted pair group method using arithmetic 2  106 and 6  107 CFU g 1 rfw (Table 2) and decreased
averages (UPGMA). for all antagonists during the time course of the experiment
about one order of magnitude, except for KS16, which
slightly increased in experiment 1 (Table 2).
Statistical analysis At the end of experiment 1, the CFU counts per gram of
lettuce rfw were not significantly different for the antago-
The STATISTICA program (StatSoft Inc., Tulsa, OK) was used
nists RU47, KF36, KS16, KS74 and KS90 (Table 2). Compar-
for the statistical analysis. The CFU data of bacterial counts
able results were obtained in experiment 2, which was
per gram rfw were logarithmically (log10) transformed
performed only with four of these isolates (RU47, KF36,
before statistical analysis. The data on inoculants’ densities,
KS16 and KS74). Despite their good ability to colonize the
plant shoot and root dry weight were analyzed after ANOVA
rhizosphere, the in vitro antagonists KS70, AFNS31 and
using Tukey’s test procedure (HSD) with P = 0.05.
RN86 failed to control the pathogen in experiment 1.

FEMS Microbiol Ecol 69 (2009) 62–74

c 2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
66 M.F. Adesina et al.

R. solani AG1-IB R. solani AG2 R. solani AG3 F. oxysporum Protease Glucanase Cellulase Chitinase Siderophore 2,4-DAPG fresh root weight

Strain code, KS and KF strains isolated on Kings’B from Swedish and French soil; AFNS strain isolated on AGS from French soil; RN and RU strains isolated on R2A from the Netherlands and the United
Almost all plants died due to high disease severity by

Kingdom, respectively (Adesina et al., 2007). Inhibition zones: 1, mycelia die back and inhibition zone of 1.0–2.9 mm; 11, inhibition zone of 3.0–5.9 mm; 111, inhibition zone of 6.0 mm and more;
Log10CFU g 1 of
R. solani infection and thus the colonization density could

4.94  0.36
4.29  0.30
3.65  1.03
4.14  0.05
4.63  0.42
4.17  0.75
3.43  0.71
4.07  0.25

–, tests conducted in this study (otherwise: Adesina et al., 2007); DAPG, detection of the phlD gene by PCR-Southern blot hybridization (M.F. Adesina et al. in preparation); ND, not determined.
not be followed until the end of experiment 1 for these
In experiment 3, the ability of isolate RU47 to colonize
the vascular tissue of lettuce leaves and roots was deter-
mined 7 weeks after sowing by dilution plating of the

suspension of surface-sterilized macerated roots and leaves

from lettuce plants inoculated with RU47. We observed no
Table 1. In vitro characterization of eight selected antagonists and rhizosphere competence (CFU per gram fresh root weight) in prescreening greenhouse experiment

colony growth on the plates, suggesting that the strain did


not grow endophytically.


Suppression of bottom rot disease by

introduced bacterial antagonists

At the end of each experiment, a total of 24 plants per

treatment (six replicates per treatment and four plants
per replicate) were used to evaluate the ability of the
inoculated antagonists to suppress bottom rot disease
Hydrolytic enzymes

caused by R. solani AG1-IB on lettuce plants. Evaluation

of the pathogen effect on lettuce was based on the number
of surviving plants and the SDW compared with the Ctrl

In experiment 1, disease severity and pathogen pressure

were high as evidenced by the high numbers of dead plants
(23 of 24 plants) and a high reduction in the shoot biomass
in the Ctrl1Rs treatment only (Table 3). However, also in



the treatments inoculated with the strong in vitro antago-

nists KS90 and KS70, as well as treatments inoculated with
the medium and weak antagonists RN86 and AFNS31,


respectively (Table 1), none or low numbers of surviving



plants and significantly reduced dry shoot biomass were

found (Table 3). This indicates a high disease pressure of the
R. solani AG1-IB strain used in this study on lettuce plants
under the given cultivation conditions. In experiment 1,

high numbers of surviving plants and a shoot biomass

Antifungal activity

comparable to those from healthy Ctrl plants were obtained

for plants inoculated with isolates RU47, KF36, KS16
and KS74 (Table 3). Based on these findings, experiments 2


and 3 were repeated only with these strains. Despite the


high level of disease severity caused by the pathogen,

16S rRNA gene partial sequencing

the highest number of surviving plants was found in






treatments inoculated with RU47 in all experiments

(Table 3), indicating a consistent and strong antagonistic
Pseudomonas fluorescens

activity of this bacterium towards R. solani AG1-IB.

Serratia marcescens

Although KF36 had a similar disease-suppressive effect as

RU47 in experiments 1 and 2, its suppression of the
P. fluorescens

P. fluorescens
P. fluorescens

P. cannabina

pathogen was inconsistent in the four experiments per-

Closest hit

P. jessenii
P. fulgida

formed. In fact, none or one surviving plant was recovered

, no activity.

from treatments inoculated with KF36 in experiments 3 and

4 (Table 3). In comparison with RU47 and KF36, strains

KS16 and KS74 were less efficient in controlling R. solani


AG1-IB in experiments 1 and 2.

c 2009 Federation of European Microbiological Societies FEMS Microbiol Ecol 69 (2009) 62–74
Published by Blackwell Publishing Ltd. All rights reserved
Biocontrol of Rhizoctonia solani and rhizosphere competence 67

Effects of bacterial antagonist on the growth of the pathogen. The shoot and root dry weight obtained for
lettuce plants plants inoculated was similar to the healthy Ctrl plants.
The effects of RU47, KF36, KS16 and KS74 (which showed
significant disease suppression in experiment 1) on lettuce Treatment effects on indigenous microbial
growth were evaluated in experiment 3. The effects of the communities
inoculants on plant growth were determined in treatments
inoculated with each bacterial antagonist without adding The effect of the pathogen R. solani1-IB and of the inoculant
strain P. jessenii RU47 on the relative abundance of domi-
nant bacterial, fungal and Pseudomonas populations in the
rhizosphere of lettuce plants was investigated using DGGE
Table 2. Colonization density of bacterial antagonists on lettuce ‘Tizian’ analysis of 16S rRNA, 18S rRNA or gacA gene fragments
(log10 CFU g 1 of fresh root weight) cultivated in growth chamber at amplified from TC-DNA. DGGE profiles were compared
20/15 1C 3, 5 and 7 weeks after sowing (WAS) in three independent
between three replicate rhizosphere samples from the non-
experiments (1, 2 and 3)
inoculated healthy control plants (Ctrl), plants grown with
Experiments 3 WAS 5 WAS 7 WAS R. solani (Ctrl1Rs) and plants with a combined inoculation
Experiment 1 of RU47 and R. solani (RU471Rs) at three sampling times
RU471Rs 7.17  0.04 cb 6.72  0.15 a 5.92  0.12 cb (3, 5 and 7 weeks after sowing). Although DGGE analyses
KF361Rs 7.57  0.27 c 6.55  0.22 a 6.63  0.12 c were performed for experiments 1, 2 and 3, only the data for
KS161Rs 6.39  0.37 ab 6.40  0.55 a 6.58  0.19 c
experiment 3 are presented here.
KS741Rs 6.57  0.26 ab 6.46  0.13 a 5.82  0.23 cb
KS901Rs 6.52  0.08 ab 6.81  0.66 a 5.95  0.39 cb
The bacterial DGGE profiles showed complex patterns
KS701Rs 6.20  0.53 a 6.21  0.58 a 4.75  0.39 a indicating many equally abundant bacterial populations.
AFNS311Rs 6.85  0.38 ab 6.15  0.33 a 5.53  0.42 ab Detection of strain RU47 in the bacterial rhizosphere
RN861Rs 6.68  0.02 ND ND patterns of inoculated plants was impossible due to the
Experiment 2 presence of a band with identical electrophoretic mobility as
RU471Rs 6.19  0.38 a 5.00  0.33 a 4.88  0.49 a the 16S rRNA gene fragment amplified from RU47 in the
KF361Rs 6.55  0.30 a 5.93  0.21 b 5.54  0.44 a
replicates of Ctrl and Ctrl1Rs treatments (Fig. 1a and b).
KS161Rs 6.53  0.07 a 5.48  0.35 ab 5.45  0.24 a
KS741Rs 5.86  0.47 a 4.96  0.38 a 4.92  0.33 a
Seven weeks after sowing, this band was much less intense in
Experiment 3 the replicates of RU471Rs (Fig. 1b). Three weeks after
RU471Rs 5.82  0.06 a 5.24  0.27 5.62  0.22 sowing, a clear effect of the seed inoculation could be
KF361Rs 5.92  0.06 a ND ND detected (data not shown). Cluster analysis (UPGMA of
KS161Rs 5.22  0.22 a ND ND Pearson’s correlation indices) of the bacterial DGGE profiles
KS741Rs 6.09  0.28 a ND ND showed that the bacterial patterns of rhizosphere samples
CFU of the same experiment followed by the same letter are not taken 5 weeks after sowing (2 weeks after transplanting)
significantly different according to Tukey’s test (P = 0.05). displayed a higher degree of variability between replicates,
ND, parameter not determined due to the death of nearly all plants, thus and the clustering of the treatments was less pronounced
no surviving plants for sampling. (Fig. 1a). Seven weeks after sowing, the separation of the

Table 3. Biological control effect of bacterial antagonists toward Rhizoctonia solani after seed and plant inoculation of lettuce ‘Tizian’ cultivated in
growth chamber at 20/15 1C for 7 weeks on number of dead plants (DP, from 24 plants) and shoot dry weight (SDW)
Experiment 1 Experiment 2 Experiment 3 Experiment 4

Treatments DP SDW (g per plant) SD DP SDW (g per plant) SD DP SDW (g per plant) SD DP SDW (g per plant) SD
Ctrl 0 5.2 a 0.4 0 3.9 a 0.2 0 2.8 a 0.1 0 1.6 a 0.05
Ctrl1Rs 23 0.9 bc 0.5 13 2.2 b 0.9 24 0.6 b 0.1 24 0.2 b 0.03
RU471Rs 0 4.7 a 0.2 0 3.5 ac 0.1 7 1.9 c 0.5 3 1.3 c 0.14
KF361Rs 0 4.9 a 0.6 0 3.4 ac 0.2 23 0.6 b 0.1 24 0.2 b 0.03
KS161Rs 1 4.1 a 0.5 5 2.8 bc 0.6 22 0.6 b 0.3
KS741Rs 4 3.9 a 0.7 3 3.0 ab 0.5 23 0.7 b 0.3
KS901Rs 11 2.2 c 1.2
KS701Rs 21 1.4 bc 1.0
AFNS311Rs 21 1.6 bc 0.8
RN861Rs 24 0.6 b 0.1

Dry weight followed by the same letter is not significantly different according to Tukey’s test (P = 0.05). DP, dead plant; SD, standard deviation.

FEMS Microbiol Ecol 69 (2009) 62–74

c 2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
68 M.F. Adesina et al.

Fig. 1. Comparison among DGGE fingerprints of bacterial 16S rRNA gene fragments amplified from community DNA extracts obtained from the
rhizosphere of lettuce plants without inoculation (Ctrl), with Rhizoctonia solani inoculation alone (Ctrl1Rs) and with combined inoculation of RU47
(seed and young plant inoculation) and R. solani (RU471Rs) at (a) 5 weeks after sowing and (b) 7 weeks after sowing. Beside each gel, the
corresponding UPGMA dendrogram based on Pearson’s correlation indices is given. A, B and C are the three independent replicates per treatment.

cluster formed by the fingerprints of the Ctrl1Rs replicates the antagonist 3 and 5 weeks after sowing (data not shown),
was more pronounced [ o 30% similarity to the Ctrl and whereas 7 weeks after sowing, the detection of RU47 was
RU471Rs treatments (Fig. 1b)]. The replicates of the not possible because a band with similar electrophoretic
RU471Rs and Ctrl treatments formed a joint cluster. mobility as the 16S rRNA gene fragment amplified from
Analysis of the Pseudomonas community pattern in the RU47 was detected in all treatments (data not shown). In
rhizosphere of lettuce plants showed a band with the same contrast to the bacterial community profiles, Pseudomonas
mobility as RU47 only in treatments inoculated with rhizosphere patterns of lettuce plants were less complex,

c 2009 Federation of European Microbiological Societies FEMS Microbiol Ecol 69 (2009) 62–74
Published by Blackwell Publishing Ltd. All rights reserved
Biocontrol of Rhizoctonia solani and rhizosphere competence 69

Fig. 2. Comparison among DGGE fingerprints

of Pseudomonas-specific gacA gene fragments
amplified from community DNA extracts
obtained from the rhizosphere of lettuce plants
without inoculation (Ctrl), with Rhizoctonia
solani inoculation alone (Ctrl1Rs) and with
combined inoculation of RU47 (seed and young
plant inoculation) and R. solani (RU471Rs) at
(a) 5 weeks after sowing and (b) 7 weeks after
sowing. Beside each gel, the corresponding
UPGMA dendrogram based on Pearson’s
correlation indices is given. A, B and C are the
three independent replicates per treatment.

with fewer numbers of bands. Aside from a band that was among all treatments. However, the fingerprints of the
pronounced in the fingerprints of the Ctrl and RU471Rs RU471Rs treatment clearly clustered separately from the
samples taken 7 weeks after sowing, the banding patterns replicates of the Ctrl1Rs and Ctrl treatments most likely
did not markedly differ at each sampling time for all due to the presence of a strong band with the same
treatments. UPGMA analysis of the Pseudomonas DGGE electrophoretic mobility as RU47. All gacA fingerprints of
banding patterns confirmed that replicates of all treatments the samples taken 7 weeks after sowing displayed a high
shared a high similarity (4 80%) at all sampling times. similarity, although treatment-dependent clustering was
Overall, a rather low degree of variability was observed observed.
among treatments, and the diversity of this group was The fungal fingerprints for rhizosphere samples taken 5
almost not affected by inoculation with RU47 (data not weeks after sowing (Fig. 3a) revealed a high similarity of the
shown). Ctrl and RU471Rs treatments with treatment-dependent
The DGGE profiles of the Pseudomonas-specific gacA separate clusters. In contrast, the samples of the Ctrl1Rs
gene fragments displayed a higher number of bands, indi- treatments were variable and two of the three replicates of
cating a better separation of Pseudomonas populations Ctrl1Rs shared o 25% with the fingerprints of all other
(Fig. 2a and b) as compared with Pseudomonas 16S rRNA samples. This trend became even more pronounced 7 weeks
DGGE profiles. A band with the mobility of RU47 was after sowing. All replicates of Ctrl1Rs treatment formed a
detected only in the RU471Rs treatments, and this band separate cluster with o 20% similarity to the fingerprints of
was clearly separated from a dominant band that was the RU471Rs and Ctrl treatment. Several bands (Fig. 3b,
observed in the fingerprints of all treatments (Fig. 2a and bands a, b, c d) were detected in the fingerprints of these
b). Although this dominant band was less well separated treatments that were not found in the patterns of the
from the RU47 band in the gacA fingerprints of rhizosphere Ctrl1Rs treatments. Obviously, the inoculation with
communities sampled 7 weeks after sowing, the inoculant R. solani AG1-IB had a major impact on the fungal
strain was still detectable at that time point for all replicates fingerprints. In contrast, the fingerprints of the RU471Rs
of RU471Rs (Fig. 2a). Also, the gacA fingerprints of samples treatment displayed a high similarity to the noninoculated
taken 5 weeks after sowing shared a high similarity (4 60%) control, indicating no major effect in this treatment on the

FEMS Microbiol Ecol 69 (2009) 62–74

c 2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
70 M.F. Adesina et al.

Fig. 3. Comparison among DGGE fingerprints of fungal 18S rRNA gene fragments amplified from community DNA extracts obtained from the
rhizosphere of lettuce plants without inoculation (Ctrl), with Rhizoctonia solani inoculation alone (Ctrl1Rs) and with combined inoculation of RU47
(seed and young plant inoculation) and R. solani (RU471Rs) at (a) 5 weeks after sowing and (b) 7 weeks after sowing. Beside each gel, the
corresponding UPGMA dendrogram based on Pearson’s correlation indices is given. A, B and C are the three independent replicates per treatment. The
bands indicated as circled a, b, c and d are bands that are common to the replicates of the Ctrl and the RU47+Rs plants.

c 2009 Federation of European Microbiological Societies FEMS Microbiol Ecol 69 (2009) 62–74
Published by Blackwell Publishing Ltd. All rights reserved
Biocontrol of Rhizoctonia solani and rhizosphere competence 71

relative abundance of dominant fungal ribotypes. The displayed strong in vitro antagonistic activity against
DGGE profiles of fungal communities revealed that a band R. solani AG1-IB.
with electrophoretic mobility corresponding to that of the In the growth chamber experiments, the bacterial antago-
18S rRNA gene fragment amplified from R. solani was much nists were applied by seed inoculation and plant drenching.
stronger in the Ctrl1Rs treatment than in the RU471Rs Drenching of young lettuce plants with a bacterial cell
treatment (Fig. 3a and b), indicating an increased relative suspension can easily be carried out before transplanting
abundance of R. solani in the lettuce rhizosphere of the lettuce seedlings into the field. The CFU counts of the
Ctrl1Rs treatment. inoculant strains per gram of rfw determined 3 weeks after
sowing in three independent growth chamber experiments
were much higher than the counts obtained in the pre-
Discussion screening greenhouse experiment. The reasons for this
In this study, in vitro antagonists were tested in growth difference might be that the soil composition and availabil-
chamber experiments with lettuce artificially inoculated ity of nutrients were different in the soils used. The ratio of
with R. solani AG1-IB in order to assess their potential to sand to substrate of the soil used for the greenhouse
colonize lettuce roots and to suppress R. solani AG1-IB- prescreening experiment was 80 : 20 in contrast to the soil
based disease symptoms and effect on plant growth. Inter- used for the growth chamber experiments, where the ratio
estingly, only one strain (P. jessenii RU47) with weak in vitro was 50 : 50. This observation might point to an influence of
antagonistic activity against R. solani AG1-1B (Table 1) the soil on the ability of the inoculant strains to colonize the
displayed efficient and consistent disease suppression rhizosphere of lettuce. Maloney et al. (1997) found that the
throughout the four independent growth chamber experi- availability of carbon and nitrogen affected the rhizobacter-
ments. These findings confirm previous observations that in ial population. The ability of inoculant strains to colonize
vitro inhibition does not necessarily correlate with in vivo the root system after seed inoculation or root inoculation
performance (Milus & Rothrock, 1997; Schottel et al., 2001; has been pinpointed as a key factor for successful biocontrol
Faltin et al., 2004; Gravel et al., 2005). by many authors (Bloemberg & Lugtenberg, 2001; Haas &
Seven of the eight strains selected were previously as- Défago, 2005). All strains showed rather good seed and root
signed to Pseudomonas by 16S rRNA gene sequencing colonization as most inoculants established at cell densities
(Adesina et al., 2007), and for all of them proteolytic activity 4 106 g 1 rfw 3 weeks after sowing. In the case of P. fulgida
and siderophore production was detected on plates. Four KS70, which displayed strong in vitro antagonistic activity
Pseudomonas fluorescens strains (KF36, KS16, KS74 and towards a range of fungi and bacteria, less efficient coloniza-
KS90), which displayed strong in vitro antagonistic activity tion might have contributed to the failure to control the
towards R. solani AG1-IB, carried the phlD gene involved in pathogen AG1-IB in the growth chamber experiment. How-
2,4-diacetylphloroglucinol (2,4-DAPG) biosynthesis (M.F. ever, P. fluorescens KS90 and S. marcescens AFNS31 also
Adesina et al., unpublished data), suggesting that the strains failed to suppress bottom rot of R. solani, but nevertheless
might produce 2,4-DAPG. The important role of 2,4-DAPG they colonized lettuce roots at the same or similar popula-
production in efficient biocontrol was demonstrated re- tion densities as the biocontrol-efficient strain RU47. This
cently (Haas & Défago, 2005; Rezzonico et al., 2007). In implies that the inability of these isolates to suppress disease
growth chamber experiment 1 all potentially 2,4-DAPG- was not due to insufficient root colonization, but may have
producing P. fluorescens strains, except for KS90, showed resulted from insufficient production of antifungal metabo-
good disease suppression. Although the strong in vitro lites, different colonization patterns, for example the colo-
antagonist P. fluorescens KS90 carried the phlD gene and its nization of the strains at locations different from the
CFU counts per gram of rfw were not significantly different infectious site of the pathogen or insufficient cell density of
from the P. fluorescens strains KS16 and KS74, the disease the antagonists at the infectious site or differences in the
control by this strain was much less efficient. However, all plant response. However, because the colonization patterns
three other phlD-carrying P. fluorescens strains inoculated in were not investigated in this study, it cannot be excluded
experiment 3 failed to control the disease. The reasons for that differences in colonization patterns between the strains
the variability observed are not clear. Differences in the existed. Using gfp-tagged inoculant strains, several studies
expression of genes involved in antibiotic production due to reported heterogeneous colonization patterns and the ques-
lower cell densities in experiment 3 in comparison with tion of what triggers the colonization of some sites and not
experiment 1 (Table 2) in combination with high pathogen of others was raised (Gamalero et al., 2003; Götz et al.,
pressure might be discussed. Four out of the eight in vitro 2006). In contrast to studies performed under field condi-
antagonists of R. solani AG1-IB selected in our study failed tions on the control of R. solani by means of bacterial
to efficiently control the pathogen in the growth chamber antagonists (Grosch et al., 2005; Scherwinski et al., 2008),
experiment 1 (Table 3), although two of these four isolates the growth chamber experiments were performed under

FEMS Microbiol Ecol 69 (2009) 62–74

c2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
72 M.F. Adesina et al.

well-controlled conditions and with an extremely high presence of R. solani AG1-IB and the inoculation with RU47
pathogen pressure. A variation still existed between the had almost no effect on the Pseudomonas group and that the
experiments (Tables 2 and 3). The major variable factor gacA DGGE fingerprints of all treatments, despite a treat-
was the growth of lettuce (Ctrl). In each of the independent ment-dependent clustering, had high similarity among the
growth chamber experiments, the Ctrl1Rs and RU471Rs treatments. This observation contradicts the hypothesis that
treatments were compared with the control. The variability organisms that are closely related to the BCA itself are most
in the lettuce growth might be due to different factors. The likely to be affected by the BCA due to competition for the
experiments were performed in the time period of c. 1 year same niches and resources (Winding et al., 2004; Götz et al.,
in the same chamber and thus it cannot be excluded that 2006). Moreover, the band patterns of the gacA-based
aging of the lamps resulted in less plant growth. The Pseudomonas community revealed remarkable diversity of
cultivation in the growth chamber started only after trans- the gacA gene in the rhizosphere of lettuce plant originating
planting at the two- to three-leaf stage, and slightly varying from all treatments. As found in this study, a better resolu-
conditions in the greenhouse might have also contributed to tion of gacA-based Pseudomonas community fingerprints
the decreased lettuce growth in experiment 2 and even more than of 16S-based Pseudomonas community fingerprints
pronounced in experiment 3. The reduced SDW determined was of reported by Costa et al. (2007).
paralleled a tendency toward decreased CFU per rfw 3 weeks In conclusion, out of the eight in vitro antagonists
after seed inoculation. Despite the variability in the growth investigated in this study, we found only one promising
of lettuce plants and CFU per rfw determined 3 weeks after strain: P. jessenii RU47. For this strain, in vitro proteolytic
seed inoculation, the biocontrol efficiency of strain RU47 activity and siderophore production were observed, while
was consistent. However, these factors might have contrib- chitinolytic, glucanolytic and cellulolytic activities, and
uted to the variable control by the phlD carrying P. fluor- genes encoding antibiotics such as 2,4-diacetylphloroglucinol
escens strains KF36, KS16 and KS74. (2,4-DAPG), phenazine, pyrrolnitrin and pyoluteorin were
Plant inoculation with bacterial antagonists has been not detected. In vitro production of salicylic acid as side-
reported to influence microbial community structure as rophores by several resistance-inducing bacteria under low-
determined by culture-independent methods that rely on iron conditions has been reported, and its role in the
amplification of rRNA gene fragments from rhizosphere induced-systemic resistance (ISR) elicitation process was
DNA extracts such as PCR-based DGGE (Götz et al., 2006). demonstrated in the case of Pseudomonas aeruginosa
In other studies, no or only transient changes were observed KMPCH (de Meyer et al., 1999). Hence, siderophore pro-
(Lottmann et al., 2000; Scherwinski et al., 2008). In the duction has been suggested to trigger the ISR signal pathway
present study, PCR-DGGE analysis enabled us to compare in plants (Audenaert et al., 2002; van Loon & Bakker, 2005).
the bacterial and fungal community profiles of the three Considering the strong and consistent suppression of the
different treatments and thus to evaluate the effect of the pathogen at very high disease severity by strain RU47 in vivo
pathogen R. solani AG1-IB in the presence or absence of on lettuce, which is contrary to the weak and rather
P. jessenii RU47 on the rhizosphere microbiota. We could insignificant direct inhibition of R. solani AG1-IB by this
demonstrate that the pathogen R. solani AG1-IB influenced strain in vitro and the in vitro production of siderophores by
not only the fungal but also the bacterial community strain RU47, it is assumed that induction of systemic
patterns. The effects of R. solani AG1-IB became more resistance in lettuce is the possible mechanism by which
pronounced during the experiment for both fungi and strain RU47 could protect lettuce plants against R. solani
bacteria. In the presence of RU47, this effect was less strong AG1-IB. Nonetheless, its exact mechanism still remains
and the fingerprints of the RU471Rs and Ctrl treatment unclear and its explanation needs future investigation.
formed a joint cluster for bacterial and fungal fingerprints. Strain RU47 did not promote shoot or root biomass of
However, the detection of RU47 was hampered in the lettuce compared with the noninoculated healthy plant in
bacterial community patterns due to the complexity of these the absence of the pathogen. On the basis of this observa-
patterns and the presence of ribotypes, which have similar tion, we suggest that for biological application under the
electrophoretic mobility as RU47. The complexity of the conditions where the strain was tested, it can only be used to
DGGE patterns was reduced with the use of group-specific control bottom rot disease in a soil infested with the
primers, thus enhancing the resolution for the Pseudomonas pathogen and not for a growth-promoting purpose under
group analyzed by DGGE. The gacA-based Pseudomonas noninfested conditions. However, future experiments under
DGGE profiles revealed that RU47 successfully established, field conditions and in different soil types have to show
as a band corresponding to the electrophoretic mobility to whether the RU47 conferred control effect is specific for R.
RU47 was found only in the rhizosphere patterns of lettuce solani AG1-IB and specific for lettuce plants or whether
of the RU471Rs treatment in the gacA-based Pseudomonas strain RU47 can also provide protection of lettuce plants
community patterns. Furthermore, we observed that the against other pathogens.

c 2009 Federation of European Microbiological Societies FEMS Microbiol Ecol 69 (2009) 62–74
Published by Blackwell Publishing Ltd. All rights reserved
Biocontrol of Rhizoctonia solani and rhizosphere competence 73

Acknowledgements Gomes NCM, Kosheleva IA, Abraham WR & Smalla K (2005)

Effects of the inoculant strain Pseudomonas putida KT2442
This work was supported by grants from DAAD and Gisela
(pNF142) and of naphthalene contamination on the soil
und Hermann Stegemann-Stiftung to M.F.A., by the
bacterial community. FEMS Microbiol Ecol 54: 21–33.
EU BIOTECH project METACONTROL and the project
González Garcı́a V, Portal Onco MA & Rubio Susan V (2006)
BRA05/021. Furthermore, the support of Angelika Fandrey, Review. Biology and systematics of the form genus
Guo-Chun Ding and Ilse-Marie Jungkurth is acknowledged. Rhizoctonia. Span J Agric Res 4: 55–79.
Götz M, Gomes NCM, Dratwinski A, Costa R, Berg G, Peixoto R,
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