Professional Documents
Culture Documents
Rhizoctonia Solani and Lettuce Biocontr and Soil Comunity
Rhizoctonia Solani and Lettuce Biocontr and Soil Comunity
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-
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
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
FEMS Microbiol Ecol 69 (2009) 62–74 c2009 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
isolates.
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
ND
suspension of surface-sterilized macerated roots and leaves
1
1
1
1
from lettuce plants inoculated with RU47. We observed no
Prescreening
Table 1. In vitro characterization of eight selected antagonists and rhizosphere competence (CFU per gram fresh root weight) in prescreening greenhouse experiment
1
1
1
1
1
111
111
1
1
1
11
100
100
98
99
99
99
%
P. fluorescens
P. fluorescens
P. cannabina
P. jessenii
P. fulgida
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
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.
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
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 c2009 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
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
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
FEMS Microbiol Ecol 69 (2009) 62–74 c2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
74 M.F. Adesina et al.
on the diversity of soil and rhizosphere bacteria and fungi. Schottel JL, Shimizu K & Kinkel LL (2001) Relationship of in vitro
Plant Soil 266: 23–29. pathogen inhibition and soil colonization to potato scab
Milus EA & Rothrock CS (1997) Efficacy of bacterial seed biocontrol by antagonistic Streptomyces spp. Biol Control 20:
treatments for controlling Pythium root rot of winter wheat. 102–112.
Plant Dis 81: 180–184. Smit E, Leeflang P, Glandorf B, van Elsas JD & Wernars K (1999)
Ogoshi A (1987) Ecology and pathogenicity of anastomosis and Analysis of fungal diversity in the wheat rhizosphere by
intraspecific groups of Rhizoctonia solani Kühn. Annu Rev sequencing of cloned PCR-amplified genes encoding 18S
Phytopathol 25: 125–143. rRNA and temperature gradient gel electrophoresis. Appl
Ogoshi A (1996) The genus Rhizoctonia. Rhizoctonia Species: Environ Microb 65: 2614–2621.
Taxonomy, Molecular Biology, Ecology, Pathology and Disease Sweetingham MW (1996) Integrated control of Rhizoctonia
Control (Sneh B, Jabaji-Hare S, Neate S & Dijst G, eds), species. Rhizoctonia Species: Taxonomy, Molecular Biology,
pp. 1–9. Kluwer Academic Publishers, Dordrecht, the Ecology, Pathology and Disease Control (Sneh B, Jabaji-Hare S,
Netherlands. Neate S & Dijst G, eds), pp. 549–558. Kluwer Academic
Raaijmakers JM & Weller DM (2001) Exploiting genotypic Publishers, Dordrecht, the Netherlands.
diversity of 2,4-diacetylphloroglucinol-producing Pseudomonas Vainio EJ & Hantula J (2000) Direct analysis of wood-inhabiting
spp.: characterization of superior root-colonizing P. fluorescens fungi using denaturing gradient gel electrophoresis of
strain Q8r1-96. Appl Environ Microb 67: 2545–2554. amplified ribosomal DNA. Mycol Res 104: 927–936.
c 2009 Federation of European Microbiological Societies FEMS Microbiol Ecol 69 (2009) 62–74
Published by Blackwell Publishing Ltd. All rights reserved