In Vitro Antagonists of Rhizoctonia Solani Tested on Lettuce Rhizosphere Competence, Bio Control Efficiency and Rhizosphere Microbial Community Response Research Article 2009 FEMS

8/8/2019 In Vitro Antagonists of Rhizoctonia Solani Tested on Lettuce Rhizosphere Competence, Bio Control Efficiency and Rh

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

In vitroantagonists of Rhizoctonia solanitested on lettuce:

rhizosphere competence, biocontrol eciency and rhizosphere

microbial community response

Modupe F. Adesina1, Rita Grosch2, Antje Lembke1, Tzenko D. Vatchev3 & Kornelia Smalla1

1Julius K uhn-Institut Federal Research Centre for Cultivated Plants (JKI), Braunschweig, Germany; 2Institute for Vegetables and Ornamental Crops

(IGZ), Grobeeren, Germany; and 3Department of Plant Pathology and Immunology, Plant Protection Institute, Sofia, Bulgaria

Correspondence: Kornelia Smalla, Julius

K uhn-Institut Federal Research Centre for

Cultivated Plants (JKI), Messeweg 11/12,

D-38104 Braunschweig, Germany. Tel.:149

531 299 3814; fax:149 531 299 3006;

e-mail: [email protected]

Received 30 July 2008; revised 18 March 2009;

accepted 24 March 2009.

Final version published online June 2009.

DOI:10.1111/j.1574-6941.2009.00685.x

Editor: Jim Prosser

Keywords

Rhizoctonia solani; antagonists; lettuce;

rhizosphere competence; biocontrol; microbial

communities.

Abstract

The rhizosphere competence of 15 in vitro antagonists of Rhizoctonia solani was

determined 4 weeks after sowing inoculated lettuce seeds into nonsterile soil.

Based on the colonization ability determined by selective plating, eight strains were

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

antagonists colonized the rhizosphere of lettuce with CFU counts above 2 106 g1

of root fresh weight, only four isolates significantly reduced disease severity. In

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 ofPseudomonas-

specific gacA genes amplified from total community DNA confirmed that RU47

established as the dominant Pseudomonas population in the rhizosphere of

inoculated lettuce plants. Furthermore, the DGGE fingerprint revealed that

R. solani AG1-IB inoculation severely affected the bacterial and fungal community

structure in the rhizosphere of lettuce and that these effects were much less

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 resultssuggest that RU47 is a promising agent to control bottom rot of lettuce.

Introduction

Rhizoctonia solani Kuhn, the anamorph of Thanatephorus

cucumeris (Frank) Donk, is a widespread soil-borne fungus

comprising plant parasitic or saprophytic strains (Ogoshi,

1987; Gonzalez Garca et al., 2006). The species affects many

important agricultural and horticultural crops worldwide,

causing diseases such as black scurf, crown rot and bottom

rot (Ogoshi, 1996). The control of the pathogen is difficult

because of its wide host range and its ability to survive as

sclerotia under adverse environmental conditions. In prac-

tice, the control of diseases caused by R. solani relies mainly

on fungicides (Kataria & Gisi, 1996). However, increasing

concern about the health and environmental hazards asso-

ciated with the use of agrochemicals has resulted in the

search for viable alternatives. Hence, biological control

involving the use of microorganisms that can suppress or

antagonize plant pathogens is being considered as a sub-

stitute or a supplement to reduce the use of chemical

pesticides (Sweetingham, 1996; Compant et al., 2005). Over

the last decades, bacteria with the ability to suppress

R. solani have been isolated from different soils (Weller

et al., 2002; Faltin et al., 2004; Garbeva et al., 2004; Berg

et al., 2005; Adesina et al., 2007). However, many studies

have reported variabilities in the performance of biological

control agents (BCA) and lack of a correlation between in

vitro inhibition tests and the field performance of BCA

(Milus & Rothrock, 1997; Schottel et al., 2001). It is assumed

that the major contributors to this inconsistency in planta

are the inherent characteristics of the BCA such as poor root

colonization or insufficient production of antifungal meta-

bolites at the pathogen infection sites due to variable

expression of genes involved in disease suppression (Raaij-

makers & Weller, 2001; Haas & Defago, 2005; Raaijmakers

et al ., 2008). Recently, the application of molecular

DNA-based methods contributed toward an improved

FEMS Microbiol Ecol 69 (2009) 6274c 2009 Federation of European Microbiological Societies

Published by Blackwell Publishing Ltd. All rights reserved
mailto:[email protected]:[email protected]


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understanding of the ecology of BCA in the rhizosphere

where the BCA interact not only with the plant and the

pathogen but also with the indigenous microbial commu-

nity (Gotz et al., 2006; Scherwinski et al., 2008).

In this study, 15 strains were selected from a collection of

bacterial isolates with in vitro antagonistic activity towards

R. solani AG3 (the causal agent of black scurf on potato) and/or Fusarium oxysporum (which causes wilting disease in

diverse crops), which were previously isolated and character-

ized (Adesina et al., 2007). The in vitro antagonists that

originated from four disease-suppressive soils located in

France, the Netherlands, Sweden and the United Kingdom

were first screened for their ability to colonize the rhizosphere

of lettuce in nonsterile soil. Based on their colonization ability,

eight strains were selected for growth chamber experiments.

The work presented here aimed to investigate the potential of

the in vitro antagonists to suppress bottom rot disease caused

byR. solani AG1-IB on lettuce plants in vivo (growth chamber

experiments) and to study the survival and root colonization

efficiency of the antagonists by means of selective plating. The

abundance of the most promising antagonist and the response

of the indigenous fungal and bacterial communities in the

rhizosphere to plant inoculation were evaluated by cultiva-

tion-independent molecular fingerprints. This is one of the

few studies assessing not only biocontrol efficiency but also

the survival of the bacterial inoculants and effects on the

indigenous microbial communities.

Materials and methods

Bacterial and fungal strains

Fifteen out of 248 antagonists isolated from four suppressive

soils (Adesina et al., 2007) were selected on the basis of

either their (1) strong in vitro activity against R. solani AG3

(isolate Ben3; host plant, potato) or (2) activity towards

both R. solani AG3 and F. oxysporum (isolate Foln3; host

plant, flax). All bacterial antagonists used in this study were

maintained on R2A agar (Difco, Detroit, MI). Rhizoctonia

solani AG1-IB (isolate 7/3; host plant, lettuce) and AG2

(isolate W4; host plant cabbage) were obtained from the

strain collection of the Institute for Vegetables and Orna-

mental Crops, Grobeeren, Germany) and maintained on

Waksman agar containing 5 g of proteose-peptone (Merck,

Darmstadt, Germany), 10 g of glucose (Merck), 3 g of meatextract (Chemex, Munchen, Germany), 5 g of NaCl (Merck),

20 g of agar (Difco), and distilled water (to 1 L) (pH 6.8).

Generation of antibiotic-resistant mutants

To facilitate reisolation of the bacterial inoculants from the

rhizosphere, spontaneous rifampicin-resistant mutants of the

bacterial antagonists were generated by plating overnight

cultures onto R2A agar supplemented with rifampicin

(75mg mL1). The 15 rifampicin-resistant mutant strains were

tested for inhibitory activity against two highly pathogenic

isolates belonging to the R. solani anastomosis groups AG1-IB

and AG2 (Faltin et al., 2004; Grosch et al., 2004). In vitro

inhibitory activity against R. solani AG1-IB and AG2 was

determined essentially as described by Adesina et al. (2007).

The mutant strains were stored at 801

C in LuriaBertani broth (ROTH, Germany) containing 20% glycerol

supplemented with rifampicin (75 mg mL1).

Lettuce rhizosphere colonization assays

To determine the rhizosphere competence of the 15 rifam-

picin-resistant strains, rhizosphere colonization assays were

performed in nonsterile soil under greenhouse conditions.

Lettuce seeds (cultivar Tizian, Syngenta, Bad Salzuflen,

Germany) were surface sterilized in 2% sodium hypochlor-

ite (NaOCl) for 5 min, followed by three subsequent wash-

ing steps with sterile distilled water. The seeds were

pregerminated on sterile moistened filter papers in sterilePetri dishes for 2 days at room temperature. Pregerminated

lettuce seeds were soaked in a cell suspension (cell concen-

tration adjusted to c. 109 cells mL1) of each bacterial

antagonist for 1 h. Four pregerminated and inoculated seeds

per pot were sown into nonsterile potting soil [turf sub-

strate/clay granulate No. 4230, Klasmann-Deilmann GmbH,

Geeste, Germany, sieved (2-mm mesh width) and mixed

with 80% (w/w) sand]. The pots were kept in the greenhouse

at 20 1C and 30% humidity. Each treatment was replicated

three times. Four weeks after sowing, the plants were care-

fully removed from the soil and vigorously shaken. The

roots with adhering soil were placed in a Stomacher plastic

bag and processed after adding sterile saline mechanically by

Stomacher blending for 3 min at high speed. CFU of the

inoculant per gram of root fresh weight (rfw) were deter-

mined by plating serial dilutions of the rhizosphere suspen-

sions on R2A supplemented with rifampicin (75 mg mL1)

and cycloheximide (100mg mL1). The CFU were counted

after 2 days of incubation at 28 1C.

Growth chamber experiments

Based on the results of the rhizosphere colonization assays,

eight antagonistic isolates with the ability to colonize lettuce

roots at cell densities of approximately log CFU 4 4 g1 of

rfw or more were selected for the growth chamber experi-

ments. The efficacy of eight antagonistic isolates in control-

ling bottom rot disease caused by R. solani AG1-IB on

lettuce plants (cultivar Tizian) was assessed under favor-

able conditions for the pathogen in the growth chamber

(York, Mannheim, Germany; 16 h/8 h day/night cycle,

500 mmolm2 s1, 20/15 1C and 60%/80% relative humid-

ity). In experiment 1, all eight in vitro antagonists were

evaluated. Four isolates with the best disease suppression

FEMS Microbiol Ecol 69 (2009) 6274 c 2009 Federation of European Microbiological Societies


63Biocontrol of Rhizoctonia solaniand rhizosphere competence


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were included in experiments 2 and 3, while only two

isolates with the best results in experiments 1 and 2 were

assessed in experiment 4. In experiment 3, the effect on plant

growth was determined based on lettuce shoot and root dry

weight, in treatments without pathogen inoculation.

Seeds were treated with bacterial antagonists directly before

sowing. Each antagonist was grown overnight on nutrient agarsupplemented with rifampicin at 75mg mL1. Overnight-

grown colonies were resuspended in a sterile 0.3% NaCl

solution and shaken to obtain a homogeneous cell suspension.

The concentration was adjusted in a spectrophotometer to a

density corresponding to c. 109CFUmL1. Lettuce seeds were

soaked in the bacterial suspension for 1 h at room temperature.

Seeds that were incubated in sterile saline for the same time

served as a control. Seeds were germinated in a seedling tray

containing 92 holes filled with a nonsterile mixture of quartz

sand and substrate [Fruhsdorfer Einheitserde Typ P, Germany;

chemical analysis (mg per 100g): N= 75, P= 75, K= 125;

pH 5.9] at a 1 : 1 ratio (v/v). Lettuce plantlets were transferred

at the two- to three-leaf stage to pots filled with the same soil

mixture with one plant per pot. Drenching of lettuce plants

with bacterial antagonists was carried out 24 h after transplant-

ing. Each plant of the treatment inoculated with RU47 and

R. solani (RU471Rs) was drenched with 20 mL of the bacterial

suspension (107 CFUmL1) and the noninoculated healthy

control (Ctrl) and inoculated with R. solani (Ctrl1Rs) treat-

ments received 20mL of sterile saline. The pathogen was added

on barley kernels infested with the R. solani AG1-IB (isolate 7/3;

Schneider et al., 1997) 1 day after drenching the plants with

bacterial antagonists. Five kernels per lettuce seedling were

placed 1 cm deep at a distance of 2 cm from the lettuce plant.

Noninfested barley kernels were added to the control treat-ments accordingly. The pots were watered daily to maintain the

substrate moisture. Each treatment consisted of six replicates

(four pots per replicate) arranged in a randomized design. Nine

additional pots (three plants per time point and treatment)

were included for microbial analysis.

Suppression of bottom rot disease by bacterial

inoculants

Seven weeks after sowing (4 weeks after pathogen inocula-

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

on (2) the effect on the shoot dry weight (SDW). The disease

incidence was assessed by recording the number of plants

with visual symptoms or the number of dead plants. The

shoot dry weight of each lettuce plant was measured.

Root colonization by bacterial inoculants

To determine the survival and root colonization efficiency

of the inoculated antagonists, rhizosphere samples were

collected at three time points: 3, 5 and 7 weeks after sowing.

For each treatment and sampling time three symptomless

plants were used. Loosely adhering soil was removed from

the roots by shaking the plants. Five grams of roots with

adhering soil were suspended in 20 mL of sterile saline and

shaken vigorously in sterile flask containing six glass beads

(0.6 mm in diameter) on a rotary shaker for 1 h at 307 r.p.m.

Aliquots of the rhizosphere suspension were immediatelyprocessed for enumeration of the inoculated strains. Serially

diluted rhizosphere suspensions were plated on R2A med-

ium (Difco) supplemented with rifampicin (75mg mL1)

and cycloheximide (100mg mL1). The remaining rhizo-

sphere suspension was centrifuged at 13 000g for 5min.

After discarding the supernatants, the cell pellets were stored

frozen at 20 1C.

The ability of the most promising antagonist RU47 to

colonize the vascular tissue of lettuce roots and leaves was

determined at 7 weeks after sowing in experiment 3. Roots

from the inoculated plants were shaken to remove loosely

adhering soils and washed in running water. One gram of

the washed roots or leaves were soaked in 1% sodium

hypochlorite for 1 min. Surface-sterilized root or leaf sam-

ples were rinsed four times with sterile distilled water and

macerated using a sterile mortal and pestle. The suspensions

of the macerated roots or leaves were serially diluted and

plated as mentioned above.

DNA extraction from rhizosphere samples

Total community (TC)-DNA was obtained from the rhizo-

sphere pellets by means of the Bio101 extraction kit

(Q.BIOgene, Carlsbad, CA) after a harsh lysis step. The TC-

DNA was purified using the GENECLEAN Spin kit (Q.BIO-

gene). DNA yields were estimated after electrophoresis in

1% agarose gel stained with ethidium bromide under UV

light in comparison with the 1-kb gene-rulerTM DNA

ladder (Fermentas, St Leon-Rot, Germany) run on the same

agarose gel. Depending on the yield of the extracted TC-

DNA, dilutions were made with sterile milliQ water between

1 : 50 and 1 : 100 (c. 15 ng) and were used as a template for

PCR amplification of bacterial 16S rRNA gene fragments.

Undiluted TC-DNA, or 1 : 10 dilutions (c. 20ng DNA)

served as a template for PCR of fungal 18S rRNA gene

fragments.

PCR amplification of 16S and 18S rRNA and gacAgene fragments for denaturing gradient gel

electrophoresis (DGGE) analysis

To amplify the 16S rRNA gene fragments of Pseudomonas

from TC-DNA the nested PCR system described by Milling

et al. (2004) was used. Diluted amplicons obtained from the

first PCR served as templates for a second PCR using the

bacterial primers F984-GC/R1378 and PCR conditions

described by Heuer et al. (1997). To generate bacterial



64 M.F. Adesina et al.


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community fingerprints, the latter primer system and

conditions were used to directly amplify 16S rRNA gene

fragments from TC-DNA. Amplification of the Pseudomonas-

specific gacA gene fragment (575 bp) was performed

using the nested PCR approach described by Costa et al.

(2007). Amplification of 18S rRNA gene fragments

(1650 bp) was done using the primer pair NS0/EF3(Messner & Prillinger, 1995; Smit et al., 1999) in a first PCR

assay, followed by a second PCR step with the primer pair

NS1/FR1GC (White et al., 1990; Vainio & Hantula, 2000).

The PCR conditions used were described by Costa et al.

(2006).

DGGE

DGGE analysis was performed using the Dcode System

apparatus (Bio-Rad Inc., Hercules, CA). The polyacrylamide

gel was a gradient gel containing 9% (fungi) or a double

gradient gel of 69% (bacteria, Pseudomonas and gacA)acrylamide with a denaturant gradient of 1838%

(fungi) or 2658% (bacteria, Pseudomonas and gacA) of

denaturants according to Gomes et al. (2005) (where 100%

denaturants contain 7 M urea and 40% formamide). Ali-

quots of PCR samples (24 mL) were applied to the DGGE

gels, and the run was performed in 1 Tris-acetate-EDTA

buffer at 58 1C with a constant voltage of 220 V for 6 h

(bacteria, Pseudomonas, gacA) or 180 V for 18 h (fungi). The

DGGE gels were silver stained, according to Heuer et al.

(2001).

Cluster analysis of DGGE fingerprints

Fungal, bacterial, Pseudomonas and gacA-based Pseudomonas

DGGE community fingerprints were analyzed using the

software package GELCOMPAR II version 5.6 (Applied Maths,

Kortrijk, Belgium). Background was subtracted using a

rolling disk method with an intensity of 10 (relative units),

and the lanes were normalized.

A dendrogram was constructed by the Pearson correlation

index for each pair of lanes within a gel and cluster analysis

by the unweighted pair group method using arithmetic

averages (UPGMA).

Statistical analysis

The STATISTICA program (StatSoft Inc., Tulsa, OK) was used

for the statistical analysis. The CFU data of bacterial counts

per gram rfw were logarithmically (log10) transformed

before statistical analysis. The data on inoculants densities,

plant shoot and root dry weight were analyzed after ANOVA

using Tukeys test procedure (HSD) with P= 0.05.

Results

In vitro activity of antagonists and colonization

of lettuce root

To facilitate monitoring of the 15 in vitro antagonistic

strains by selective plating, rifampicin-resistant mutants

were generated for all antagonists. The ability of the 15in vitro antagonists to colonize the roots of lettuce plants

was determined 4 weeks after sowing inoculated pregermi-

nated lettuce seeds into nonsterile soil. Eight strains with

CFU counts 4 4 103 g1 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,

lettuce), AG2 (host plant, sugar beet) and AG3 (host plant:

potato) are given in Table 1. Five antagonists (KS16, KS90,

KS70, KS74 and KF36) showed strong in vitro inhibitory

activity against R. solani AG1-IB (the model pathogen used

in this study for the growth chamber experiments) withinhibition zones larger than 6 mm while strain RN86

expressed a moderate inhibitory activity of o 6mm zone

of inhibition. OnlySerratia marcescens AFNS31 and Pseudo-

monas jessenii RU47 displayed weak activity against R. solani

AG1-IB with inhibition zones o 3mm after 5 days of

incubation. Information on the identity of the strains

selected for the growth chamber experiment, their in vitro

antagonistic activity and their secondary metabolite pro-

duction is given in Table 1.

Survival of the antagonists in the rhizosphere

of lettuce plants under growth chamber

conditions

Although similar cell densities of bacterial suspension were

used for seed inoculation (c. 109 mL1), the cell densities of

antagonists on the seed surfaces showed remarkable differ-

ences and were in the range of 8 104 8 107 CFU per seed

(data not shown). However, the bacterial inoculants actively

colonized the lettuce roots. The CFU counts of the inoculant

strains determined 3 weeks after sowing ranged between

2 106 and 6 107 CFUg1 rfw (Table 2) and decreased

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).

At the end of experiment 1, the CFU counts per gram of

lettuce rfw were not significantly different for the antago-

nists RU47, KF36, KS16, KS74 and KS90 (Table 2). Compar-

able results were obtained in experiment 2, which was

performed only with four of these isolates (RU47, KF36,

KS16 and KS74). Despite their good ability to colonize the

rhizosphere, the in vitro antagonists KS70, AFNS31 and

RN86 failed to control the pathogen in experiment 1.





5/13

Almost all plants died due to high disease severity by

R. solani infection and thus the colonization density could

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 thesuspension of surface-sterilized macerated roots and leaves

from lettuce plants inoculated with RU47. We observed no

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

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

treatment.

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 werefound (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

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,

the highest number of surviving plants was found in

treatments inoculated with RU47 in all experiments

(Table 3), indicating a consistent and strong antagonistic

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

Although KF36 had a similar disease-suppressive effect as

RU47 in experiments 1 and 2, its suppression of the

pathogen was inconsistent in the four experiments per-

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

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.Table

1.

Invitrocharacterization

ofeightselectedantagonistsandrhizospherecompetence(CFUpergramfreshrootweight)in

prescreeninggreenhouseexperiment

Codes

16SrRNAgenepartialsequencing

Antifungalactivity

Hydrolyticenzym

es

Prescreening

Closesthit

%

R.

solan

iAG1-IB

R.

solan

iAG2

R.

solan

iAG3

F.oxysporum

Protease

Glucanase

Cellulase

Chitinase

Siderophore

2,4-D

APG

Log10CFUg

1

of

freshrootweight

KS16

Pseu

domonas

fluorescen

s

100

111

1

111

11

1

111

1

4.94

0.36

KS90

P.fluorescens

100

111

11

111

1

111

1

4.29

0.30

KS70

P.fulgida

98

111

11

11

1

1

111

3.65

1.03

KS74

P.fluorescens

100

111

11

111

1

1

111

1

4.14

0.05

KF36

P.fluorescens

99

111

11

111

1

111

1

4.63

0.42

AFNS31

Serratiamarcescens

100

1

1

1

1

1

1

ND

4.17

0.75

RN86

P.canna

bina

99

11

1

1

1

1

3.43

0.71

RU47

P.jessen

ii

99

1

1

1

1

1

11

4.07

0.25

Straincode,KSandKFstrainsisolatedonKingsBfromSwedishandFrenchsoil;

AFNSstrainisolatedonAGSfromFrenchsoil;R

NandRUstrainsisolatedonR2AfromtheNet

herlandsandtheUnited

Kingdom,respectively(Adesinaet

al.,2007).Inhibitionzones:1,myceliadiebackandinhibitionzoneof1.02.9mm;11,

inhib

itionzoneof3.05.9mm;111,

inhibitionzon

eof6.0mmandmore;

,noactivity.

,testsconductedinthisstudy(otherwise:Adesinaeta

l.,2007);DAPG,

detectionofthep

hlDgenebyPCR-Southernblothybrid

ization(M.F.Adesinaeta

l.inpreparation);ND,

notdetermined.





6/13

Effects of bacterial antagonist on the growth of

lettuce plants

The effects of RU47, KF36, KS16 and KS74 (which showed

significant disease suppression in experiment 1) on lettuce

growth were evaluated in experiment 3. The effects of the

inoculants on plant growth were determined in treatments

inoculated with each bacterial antagonist without adding

the pathogen. The shoot and root dry weight obtained for

plants inoculated was similar to the healthy Ctrl plants.

Treatment effects on indigenous microbial

communities

The effect of the pathogen R. solani1-IB and of the inoculantstrain 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

analysis of 16S rRNA, 18S rRNA or gacA gene fragments

amplified from TC-DNA. DGGE profiles were compared

between three replicate rhizosphere samples from the non-

inoculated healthy control plants (Ctrl), plants grown with

R. solani (Ctrl1Rs) and plants with a combined inoculation

of RU47 and R. solani (RU471Rs) at three sampling times

(3, 5 and 7 weeks after sowing). Although DGGE analyses

were performed for experiments 1, 2 and 3, only the data for

experiment 3 are presented here.The bacterial DGGE profiles showed complex patterns

indicating many equally abundant bacterial populations.

Detection of strain RU47 in the bacterial rhizosphere

patterns of inoculated plants was impossible due to the

presence of a band with identical electrophoretic mobility as

the 16S rRNA gene fragment amplified from RU47 in the

replicates of Ctrl and Ctrl1Rs treatments (Fig. 1a and b).

Seven weeks after sowing, this band was much less intense in

the replicates of RU471Rs (Fig. 1b). Three weeks after

sowing, a clear effect of the seed inoculation could be

detected (data not shown). Cluster analysis (UPGMA of

Pearsons correlation indices) of the bacterial DGGE profiles

showed that the bacterial patterns of rhizosphere samplestaken 5 weeks after sowing (2 weeks after transplanting)

displayed a higher degree of variability between replicates,

and the clustering of the treatments was less pronounced

(Fig. 1a). Seven weeks after sowing, the separation of the

Table 2. Colonization density of bacterial antagonists on lettuce Tizian

(log10 CFUg1 of fresh root weight) cultivated in growth chamber at

20/151C 3, 5 and 7 weeks after sowing (WAS) in three independent

experiments (1, 2 and 3)

Experiments 3 WAS 5 WAS 7 WAS

Experiment 1

RU471Rs 7.170.04 cb 6.720.15 a 5.920.12 cb

KF361Rs 7.570.27 c 6.550.22 a 6.630.12 c

KS161Rs 6.390.37 ab 6.400.55 a 6.580.19 c

KS741Rs 6.570.26 ab 6.460.13 a 5.820.23 cbKS901Rs 6.520.08 ab 6.810.66 a 5.950.39 cb

KS701Rs 6.200.53 a 6.210.58 a 4.750.39 a

AFNS311Rs 6.850.38 ab 6.150.33 a 5.530.42 ab

RN861Rs 6.680.02 ND ND

Experiment 2

RU471Rs 6.190.38 a 5.000.33 a 4.880.49 a

KF361Rs 6.550.30 a 5.930.21 b 5.540.44 a

KS161Rs 6.530.07 a 5.480.35 ab 5.450.24 a

KS741Rs 5.860.47 a 4.960.38 a 4.920.33 a

Experiment 3

RU471Rs 5.820.06 a 5.240.27 5.620.22

KF361Rs 5.920.06 a ND ND

KS161Rs 5.220.22 a ND ND

KS741Rs 6.090.28 a ND ND

CFU of the same experiment followed by the same letter are not

significantly different according to Tukeys test (P= 0.05).

ND, parameter not determined due to the death of nearly all plants, thus

no surviving plants for sampling.

Table 3. Biological control effect of bacterial antagonists toward Rhizoctonia solaniafter 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)

Treatments

Experiment 1 Experiment 2 Experiment 3 Experiment 4

DP SDW (g p er plant) SD DP SDW (g p er plant) SD DP SDW (g per plant) SD DP SDW (g p er 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 0.03RU471Rs 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 Tukeys test (P= 0.05). DP, dead plant; SD, standard deviation.





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cluster formed by the fingerprints of the Ctrl1Rs replicates

was more pronounced [o 30% similarity to the Ctrl and

RU471Rs treatments (Fig. 1b)]. The replicates of the

RU471Rs and Ctrl treatments formed a joint cluster.

Analysis of the Pseudomonas community pattern in the

rhizosphere of lettuce plants showed a band with the same

mobility as RU47 only in treatments inoculated with

the antagonist 3 and 5 weeks after sowing (data not shown),

whereas 7 weeks after sowing, the detection of RU47 was

not possible because a band with similar electrophoretic

mobility as the 16S rRNA gene fragment amplified from

RU47 was detected in all treatments (data not shown). In

contrast to the bacterial community profiles, Pseudomonas

rhizosphere patterns of lettuce plants were less complex,

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 Pearsons correlation indices is given. A, B and C are the three independent replicates per treatment.





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with fewer numbers of bands. Aside from a band that was

pronounced in the fingerprints of the Ctrl and RU471Rs

samples taken 7 weeks after sowing, the banding patterns

did not markedly differ at each sampling time for alltreatments. UPGMA analysis of the Pseudomonas DGGE

banding patterns confirmed that replicates of all treatments

shared a high similarity (4 80%) at all sampling times.

Overall, a rather low degree of variability was observed

among treatments, and the diversity of this group was

almost not affected by inoculation with RU47 (data not

shown).

The DGGE profiles of the Pseudomonas-specific gacA

gene fragments displayed a higher number of bands, indi-

cating a better separation of Pseudomonas populations

(Fig. 2a and b) as compared with Pseudomonas 16S rRNA

DGGE profiles. A band with the mobility of RU47 was

detected only in the RU471Rs treatments, and this band

was clearly separated from a dominant band that was

observed in the fingerprints of all treatments (Fig. 2a and

b). Although this dominant band was less well separated

from the RU47 band in the gacA fingerprints of rhizosphere

communities sampled 7 weeks after sowing, the inoculant

strain was still detectable at that time point for all replicates

of RU471Rs (Fig. 2a). Also, the gacA fingerprints of samples

taken 5 weeks after sowing shared a high similarity (4 60%)

among all treatments. However, the fingerprints of the

RU471Rs treatment clearly clustered separately from the

replicates of the Ctrl1Rs and Ctrl treatments most likely

due to the presence of a strong band with the sameelectrophoretic mobility as RU47. All gacA fingerprints of

the samples taken 7 weeks after sowing displayed a high

similarity, although treatment-dependent clustering was

observed.

The fungal fingerprints for rhizosphere samples taken 5

weeks after sowing (Fig. 3a) revealed a high similarity of the

Ctrl and RU471Rs treatments with treatment-dependent

separate clusters. In contrast, the samples of the Ctrl1Rs

treatments were variable and two of the three replicates of

Ctrl1Rs shared o 25% with the fingerprints of all other

samples. This trend became even more pronounced 7 weeks

after sowing. All replicates of Ctrl1Rs treatment formed a

separate cluster with o 20% similarity to the fingerprints of

the RU471Rs and Ctrl treatment. Several bands (Fig. 3b,

bands a, b, c d) were detected in the fingerprints of these

treatments that were not found in the patterns of the

Ctrl1Rs treatments. Obviously, the inoculation with

R. solani AG1-IB had a major impact on the fungal

fingerprints. In contrast, the fingerprints of the RU471Rs

treatment displayed a high similarity to the noninoculated

control, indicating no major effect in this treatment on the

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 Pearsons

correlation indices is given. A, B and C are the

three independent replicates per treatment.





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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 Pearsons 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.





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relative abundance of dominant fungal ribotypes. The

DGGE profiles of fungal communities revealed that a band

with electrophoretic mobility corresponding to that of the

18S rRNA gene fragment amplified from R. solani was much

stronger in the Ctrl1Rs treatment than in the RU471Rs

treatment (Fig. 3a and b), indicating an increased relative

abundance of R. solani in the lettuce rhizosphere of theCtrl1Rs treatment.

Discussion

In this study, in vitro antagonists were tested in growth

chamber experiments with lettuce artificially inoculated

with R. solani AG1-IB in order to assess their potential to

colonize lettuce roots and to suppress R. solani AG1-IB-

based disease symptoms and effect on plant growth. Inter-

estingly, only one strain (P. jessenii RU47) with weakin vitro

antagonistic activity against R. solani AG1-1B (Table 1)

displayed efficient and consistent disease suppression

throughout the four independent growth chamber experi-

ments. These findings confirm previous observations that in

vitro inhibition does not necessarily correlate with in vivo

performance (Milus & Rothrock, 1997; Schottel et al., 2001;

Faltin et al., 2004; Gravel et al., 2005).

Seven of the eight strains selected were previously as-

signed to Pseudomonas by 16S rRNA gene sequencing

(Adesina et al., 2007), and for all of them proteolytic activity

and siderophore production was detected on plates. Four

Pseudomonas fluorescens strains (KF36, KS16, KS74 and

KS90), which displayed strong in vitro antagonistic activity

towards R. solani AG1-IB, carried the phlD gene involved in

2,4-diacetylphloroglucinol (2,4-DAPG) biosynthesis (M.F.Adesina et al., unpublished data), suggesting that the strains

might produce 2,4-DAPG. The important role of 2,4-DAPG

production in efficient biocontrol was demonstrated re-

cently (Haas & Defago, 2005; Rezzonico et al., 2007). In

growth chamber experiment 1 all potentially 2,4-DAPG-

producing P. fluorescens strains, except for KS90, showed

good disease suppression. Although the strong in vitro

antagonist P. fluorescens KS90 carried the phlD gene and its

CFU counts per gram of rfw were not significantly different

from the P. fluorescens strains KS16 and KS74, the disease

control by this strain was much less efficient. However, all

three other phlD-carrying P. fluorescens strains inoculated in

experiment 3 failed to control the disease. The reasons for

the variability observed are not clear. Differences in the

expression of genes involved in antibiotic production due to

lower cell densities in experiment 3 in comparison with

experiment 1 (Table 2) in combination with high pathogen

pressure might be discussed. Four out of the eight in vitro

antagonists ofR. solani AG1-IB selected in our study failed

to efficiently control the pathogen in the growth chamber

experiment 1 (Table 3), although two of these four isolates

displayed strong in vitro antagonistic activity against

R. solani AG1-IB.

In the growth chamber experiments, the bacterial antago-

nists were applied by seed inoculation and plant drenching.

Drenching of young lettuce plants with a bacterial cell

suspension can easily be carried out before transplanting

lettuce seedlings into the field. The CFU counts of theinoculant 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-

screening greenhouse experiment. The reasons for this

difference might be that the soil composition and availabil-

ity of nutrients were different in the soils used. The ratio of

sand to substrate of the soil used for the greenhouse

prescreening experiment was 80 : 20 in contrast to the soil

used for the growth chamber experiments, where the ratio

was 50 : 50. This observation might point to an influence of

the soil on the ability of the inoculant strains to colonize the

rhizosphere of lettuce. Maloneyet al. (1997) found that the

availability of carbon and nitrogen affected the rhizobacter-

ial population. The ability of inoculant strains to colonize

the root system after seed inoculation or root inoculation

has been pinpointed as a key factor for successful biocontrol

by many authors (Bloemberg & Lugtenberg, 2001; Haas &

Defago, 2005). All strains showed rather good seed and root

colonization as most inoculants established at cell densities

4 106 g1 rfw 3 weeks after sowing. In the case of P. fulgida

KS70, which displayed strong in vitro antagonistic activity

towards a range of fungi and bacteria, less efficient coloniza-

tion might have contributed to the failure to control the

pathogen AG1-IB in the growth chamber experiment. How-

ever, P. fluorescens KS90 and S. marcescens AFNS31 alsofailed to suppress bottom rot of R. solani, but nevertheless

they colonized lettuce roots at the same or similar popula-

tion densities as the biocontrol-efficient strain RU47. This

implies that the inability of these isolates to suppress disease

was not due to insufficient root colonization, but may have

resulted from insufficient production of antifungal metabo-

lites, different colonization patterns, for example the colo-

nization of the strains at locations different from the

infectious site of the pathogen or insufficient cell density of

the antagonists at the infectious site or differences in the

plant response. However, because the colonization patterns

were not investigated in this study, it cannot be excluded

that differences in colonization patterns between the strains

existed. Using gfp-tagged inoculant strains, several studies

reported heterogeneous colonization patterns and the ques-

tion of what triggers the colonization of some sites and not

of others was raised (Gamalero et al., 2003; Gotz et al.,

2006). In contrast to studies performed under field condi-

tions on the control of R. solani by means of bacterial

antagonists (Grosch et al., 2005; Scherwinski et al., 2008),

the growth chamber experiments were performed under





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well-controlled conditions and with an extremely high

pathogen pressure. A variation still existed between the

experiments (Tables 2 and 3). The major variable factor

was the growth of lettuce (Ctrl). In each of the independent

growth chamber experiments, the Ctrl1Rs and RU471Rs

treatments were compared with the control. The variability

in the lettuce growth might be due to different factors. Theexperiments were performed in the time period of c. 1 year

in the same chamber and thus it cannot be excluded that

aging of the lamps resulted in less plant growth. The

cultivation in the growth chamber started only after trans-

planting at the two- to three-leaf stage, and slightly varying

conditions in the greenhouse might have also contributed to

the decreased lettuce growth in experiment 2 and even more

pronounced in experiment 3. The reduced SDW determined

paralleled a tendency toward decreased CFU per rfw 3 weeks

after seed inoculation. Despite the variability in the growth

of lettuce plants and CFU per rfw determined 3 weeks after

seed inoculation, the biocontrol efficiency of strain RU47

was consistent. However, these factors might have contrib-

uted to the variable control by the phlD carrying P. fluor-

escens strains KF36, KS16 and KS74.

Plant inoculation with bacterial antagonists has been

reported to influence microbial community structure as

determined by culture-independent methods that rely on

amplification of rRNA gene fragments from rhizosphere

DNA extracts such as PCR-based DGGE (Gotz et al., 2006).

In other studies, no or only transient changes were observed

(Lottmann et al., 2000; Scherwinski et al., 2008). In the

present study, PCR-DGGE analysis enabled us to compare

the bacterial and fungal community profiles of the three

different treatments and thus to evaluate the effect of thepathogen R. solani AG1-IB in the presence or absence of

P. jessenii RU47 on the rhizosphere microbiota. We could

demonstrate that the pathogen R. solani AG1-IB influenced

not only the fungal but also the bacterial community

patterns. The effects of R. solani AG1-IB became more

pronounced during the experiment for both fungi and

bacteria. In the presence of RU47, this effect was less strong

and the fingerprints of the RU471Rs and Ctrl treatment

formed a joint cluster for bacterial and fungal fingerprints.

However, the detection of RU47 was hampered in the

bacterial community patterns due to the complexity of these

patterns and the presence of ribotypes, which have similar

electrophoretic mobility as RU47. The complexity of the

DGGE patterns was reduced with the use of group-specific

primers, thus enhancing the resolution for the Pseudomonas

group analyzed by DGGE. The gacA-based Pseudomonas

DGGE profiles revealed that RU47 successfully established,

as a band corresponding to the electrophoretic mobility to

RU47 was found only in the rhizosphere patterns of lettuce

of the RU471Rs treatment in the gacA-based Pseudomonas

community patterns. Furthermore, we observed that the

presence ofR. solani AG1-IB and the inoculation with RU47

had almost no effect on the Pseudomonas group and that the

gacA DGGE fingerprints of all treatments, despite a treat-

ment-dependent clustering, had high similarity among the

treatments. This observation contradicts the hypothesis that

organisms that are closely related to the BCA itself are most

likely to be affected by the BCA due to competition for thesame niches and resources (Winding et al., 2004; Gotz et al.,

2006). Moreover, the band patterns of the gacA-based

Pseudomonas community revealed remarkable diversity of

the gacA gene in the rhizosphere of lettuce plant originating

from all treatments. As found in this study, a better resolu-

tion of gacA-based Pseudomonas community fingerprints

than of 16S-based Pseudomonas community fingerprints

was of reported by Costa et al. (2007).

In conclusion, out of the eight in vitro antagonists

investigated in this study, we found only one promising

strain: P. jessenii RU47. For this strain, in vitro proteolytic

activity and siderophore production were observed, while

chitinolytic, glucanolytic and cellulolytic activities, and

genes encoding antibiotics such as 2,4-diacetylphloroglucinol

(2,4-DAPG), phenazine, pyrrolnitrin and pyoluteorin were

not detected. In vitro production of salicylic acid as side-

rophores by several resistance-inducing bacteria under low-

iron conditions has been reported, and its role in the

induced-systemic resistance (ISR) elicitation process was

demonstrated in the case of Pseudomonas aeruginosa

KMPCH (de Meyer et al., 1999). Hence, siderophore pro-

duction has been suggested to trigger the ISR signal pathway

in plants (Audenaert et al., 2002; van Loon & Bakker, 2005).

Considering the strong and consistent suppression of the

pathogen at very high disease severity by strain RU47 in vivoon lettuce, which is contrary to the weak and rather

insignificant direct inhibition of R. solani AG1-IB by this

strain in vitro and the in vitro production of siderophores by

strain RU47, it is assumed that induction of systemic

resistance in lettuce is the possible mechanism by which

strain RU47 could protect lettuce plants against R. solani

AG1-IB. Nonetheless, its exact mechanism still remains

unclear and its explanation needs future investigation.

Strain RU47 did not promote shoot or root biomass of

lettuce compared with the noninoculated healthy plant in

the absence of the pathogen. On the basis of this observa-

tion, we suggest that for biological application under the

conditions where the strain was tested, it can only be used to

control bottom rot disease in a soil infested with the

pathogen and not for a growth-promoting purpose under

noninfested conditions. However, future experiments under

field conditions and in different soil types have to show

whether the RU47 conferred control effect is specific for R.

solani AG1-IB and specific for lettuce plants or whether

strain RU47 can also provide protection of lettuce plants

against other pathogens.





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Acknowledgements

This work was supported by grants from DAAD and Gisela

und Hermann Stegemann-Stiftung to M.F.A., by the

EU BIOTECH project METACONTROL and the project

BRA05/021. Furthermore, the support of Angelika Fandrey,

Guo-Chun Ding and Ilse-Marie Jungkurth is acknowledged.

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In Vitro Antagonists of Rhizoctonia Solani Tested on Lettuce Rhizosphere Competence, Bio Control Efficiency and Rhizosphere Microbial Community Response Research Article 2009 FEMS

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