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Applied and Environmental Microbiology, August 2000, p. 3515-3518, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Lipopolysaccharides of Rhizobium etli
Strain G12 Act in Potato Roots as an Inducing Agent of Systemic
Resistance to Infection by the Cyst Nematode Globodera
pallida
M.
Reitz,1
K.
Rudolph,2
I.
Schröder,2
S.
Hoffmann-Hergarten,1
J.
Hallmann,1 and
R.
A.
Sikora1,*
Institut für Pflanzenkrankheiten,
Phytomedizin in Bodenökosystemen, Universität Bonn, D-53115
Bonn,1 and Institut für
Pflanzenpathologie und Pflanzenschutz, Universität
Göttingen, D-37077 Göttingen,2
Germany
Received 3 December 1999/Accepted 10 April 2000
 |
ABSTRACT |
Recent studies have shown that living and heat-killed cells of the
rhizobacterium Rhizobium etli strain G12 induce in potato roots systemic resistance to infection by the potato cyst nematode Globodera pallida. To better understand the mechanisms of
induced resistance, we focused on identifying the inducing agent.
Since heat-stable bacterial surface carbohydrates such as
exopolysaccharides (EPS) and lipopolysaccharides (LPS) are essential
for recognition in the symbiotic interaction between
Rhizobium and legumes, their role in the R. etli-potato interaction was studied. EPS and LPS were extracted
from bacterial cultures, applied to potato roots, and tested for
activity as an inducer of plant resistance to the plant-parasitic
nematode. Whereas EPS did not affect G. pallida infection,
LPS reduced nematode infection significantly in concentrations as low
as 1 and 0.1 mg ml
1. Split-root experiments, guaranteeing
a spatial separation of inducing agent and challenging pathogen, showed
that soil treatments of one half of the root system with LPS resulted
in a highly significant (up to 37%) systemic induced reduction of
G. pallida infection of potato roots in the other half. The
results clearly showed that LPS of R. etli G12 act as the
inducing agent of systemic resistance in potato roots.
| |
INTRODUCTION |
Antagonistic rhizobacteria have been
repeatedly shown to be promising microorganisms for the biological
control of plant-parasitic nematodes. In a screening program, 16 bacterial isolates out of 179 isolated from root and cysts caused a
significant (>25%) reduction in Globodera pallida
penetration of potato roots (27). A 68% reduction of sugar
beet cyst nematode root invasion was obtained by application of the
rhizobacterium Pseudomonas fluorescens P523 to beet seeds
(23). Studies on a number of plant-microbe interactions showed that such antagonistic rhizobacteria can function directly by
competition and antibiosis (3) but also indirectly by
inducing systemic resistance in the plant toward soil-borne pathogens
(9, 17, 36). However, bacterial compounds which induce plant
defense mechanisms are highly variable. Enhanced defense by
Pseudomonas aeruginosa strain 7NSK2 in bean toward the
pathogenic fungus Botrytis cinerea was initiated by
bacterial salicylic acid (5). The siderophore pyoverdin of
P. fluorescens strain CHAO was involved in systemically
induced suppression of tobacco necrosis virus in tobacco
(8). In tomato and soybean leaves, lipopolysaccharides (LPS)
of incompatible pseudomonads induced resistance against challenge
inoculations by compatible bacteria (20). Induced systemic
resistance in carnation to Fusarium wilt was triggered by
heat-killed cells and purified LPS, extracted from the outer membrane
of P. fluorescens strain WCS417r (36).
Previous work demonstrated that living and heat-killed cells of
Rhizobium etli G12 induced in potato roots systemic
resistance against G. pallida infection (9, 11).
The results of these studies suggested that heat-stable surface
structures of R. etli G12 may be the inducing factors.
Surface carbohydrates of Rhizobium consist mainly of
exopolysaccharides (EPS) as additional capsular or slimy layers around
the bacterial cell and LPS, which are an integral part of the outer
membrane of the cell. Surface carbohydrates play an important role
during the recognition process in the symbiotic interaction between
Rhizobium and legumes (6, 16). Furthermore, some
authors proposed that degradation of rhizobial polysaccharides is
involved in the regulation of the plant response (19).
The objective of this investigation was to extract EPS and LPS from the
rhizobacterium R. etli G12 and to determine whether these
carbohydrates act as inducers of systemic resistance in potato roots to
G. pallida infection.
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MATERIALS AND METHODS |
Potato cultivar.
The potato cultivar Hansa, susceptible to
G. pallida, was used in all experiments. Potato tubers were
pregerminated at room temperature in the dark for 4 weeks, and sprouts
approximately 2 cm in length with adjacent tuber tissue were cut and
used for bioassays.
Bacterial inoculum.
R. etli G12 was originally
isolated from the rhizosphere of potatoes and was repeatedly shown to
suppress early root infection by the potato cyst nematode G. pallida (9, 28; M. Reitz, S. Hoffmann-Hergarten, J. Hallmann, and R. A. Sikora, submitted for
publication). The bacterium was initially identified as
Agrobacterium radiobacter but in 1998 was renamed
Rhizobium etli. The bacteria were cultured in liquid King's
medium B (KMB; pH 5.8) (13) on a rotary shaker for 36 h
at 24°C. The bacterial suspension was centrifuged at 4°C for 20 min
at 4,600 × g (Haereus Varifuge RF), and the bacterial
cells in the pellet were resuspended in sterile one-fourth-concentrated
Ringer solution (Merck). The optical cell density at 560 nm
(OD560) was adjusted with a spectral photometer to 2.0, which corresponded to cell numbers of R. etli G12 of
approximately 1.2 × 1010 CFU ml1.
EPS and LPS extraction.
For EPS extraction, R. etli G12 was cultured on 15 agar plates with KMB at 24°C. After
3 days, the growth was scraped from the agar plates, suspended in a
500-ml sterile 0.9% NaCl solution containing 5 mM EDTA, and thoroughly
stirred. After centrifugation at 4,600 × g for 20 min
at 4°C, the supernatant containing loose and bound EPS was sterile
filtered (pore size, 0.2 µm) to remove any remaining bacterial cells.
The EPS solution was dialyzed (12,000 Da; Serva) against
demineralized water for 4 days and lyophilized.
LPS of R. etli G12 were extracted from cells grown in 10 liters of KMB broth in three 3.5-liter plastic fermentors amended with
25 ppm of antifoam solution (Dow Corning antifoam emulsion; Boehringer
Ingelheim). After 36 h of fermentation, the cells were harvested
by centrifugation at 4°C for 15 min at 4,600 × g and washed three times with 0.5 M NaCl containing 5 mM EDTA to remove loose
and bound EPS. The bacterial pellet was then lyophilized (5.5 mg [dry
weight]), suspended in 50 ml of buffer L (50 mM sodium phosphate
buffer [pH 7.0], 5 mM EDTA, 0.05% sodium azide), and digested by hen
egg white lysozyme (6 mg g1 [dry weight], 50,000 U;
Sigma) at 4°C for 16 h (12). The bacterial extract
was then treated with DNase (0.3 mg g1 [dry weight],
2,000 U mg1 [solid]; Boehringer) and RNase (0.3 mg
g1 [dry weight], 37 U mg1 [solid];
Sigma) at 37°C for 30 min. Remaining protein was digested overnight
by incubation with proteinase K (0.3 mg g1 [dry
weight], 15 U mg1 [solid]; Sigma) followed by
incubation for 10 min at 60°C to denature the protein. Finally, LPS
was purified by the hot phenol-water method (40). The
aqueous LPS solution was dialyzed (12,000 Da; Serva) for 4 days
against demineralized water to remove traces of phenol and again
lyophilized. Two stock solutions of EPS and LPS were prepared: 10 mg ml
of demineralized water1 (for analysis) and 1 mg ml of
sterile one-fourth-concentrated Ringer solution1 (for bioassays).
LPS analysis.
LPS patterns were determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (14).
A 7.5-µl aliquot taken from each sample (10 mg/ml) was applied per
slot. The polyacrylamide concentration was 12.5% in the separation gel
and 6% in the stacking gel. LPS were stained by the silver-staining
procedure (34).
The concentration of 2-keto-3-deoxyoctanate (KDO), a characteristic
sugar component of the core region of bacterial LPS, was
determined
after hydrolysis (
38). Aliquots of 5, 10, 20, and
30 µl of
the sample (10 mg ml
1) were adjusted to 50 µl with
demineralized water and hydrolyzed
in acetate buffer (pH 4.4) for
2 h at 100°C. A 0.5 mM KDO (0 to
30 µl) solution was used as a
standard.
Protein contents were determined by the Bio-Rad protein assay
(catalogue no. 500-0007) (
2). Bovine serum albumin (0 to
0.5 mg/ml) was used as a
standard.
Nematode inoculum.
The potato cyst nematode G. pallida was originally isolated from a field population near Bonn
and was multiplied continually on potato roots (9). Cysts
for experiments were extracted from soil by the wet sieve decanting
technique (1). The nematode inoculum was prepared by placing
25 cysts in a pouch made of 100-µm-mesh gauze sandwiched in a slide
frame (26, 33). The total inoculum was 1,500 eggs and
juveniles per 100 g of soil, which represents the economic
threshold level on potato.
Bioassays. (i) Root dipping.
Pregerminated potato tubers
were placed in a plastic box containing heat-sterilized sand and were
incubated in a climatic chamber at 21°C and a photoperiod of 16 h. After 5 days, the plantlets were removed and the root system of each
plant was thoroughly rinsed with tap water; the tubers were then dipped
for 2 min in a 20-ml suspension of either R. etli
(OD560 = 2.0), EPS (1 mg ml1), or LPS
(0.1 or 1 mg ml1). Sterile one-fourth-concentrated Ringer
solution (Merck) was used as a control. Treated potato plants were
transferred individually into plastic pots (8 cm in diameter) filled
with a heat-sterilized mixture of sand and field soil (1:1) and were
grown under the same conditions as described above. Two days after
bacterial colonization, the plants were inoculated with nematodes by
inserting one slide frame containing the 25 cysts into the soil about 2 cm from the root. Each treatment was performed in three independent
experiments with nine replicates per treatment.
(ii) Split-root system.
The split-root (three-pot) system
allows inoculation of the bacterium and cyst nematode at separate
locations on the root system (9). Three plastic pots
(diameter of 8 cm) were filled with a 1:1 mixture of sterilized sand
and field soil. Pregerminated potato tubers were planted in the upper
pot to allow the root system to grow through the two openings in the
bottom of the upper pot and to spread to the lower two pots. After 3 weeks, plants were inoculated with living bacterial cells or with the
LPS solution by pipetting 2.5 ml of each suspension into the soil of
one side of the split-root system. One-fourth-concentrated Ringer
solution (Merck) served as control treatment. After 2 days, the other
side of the split-root system was inoculated with nematodes by
inserting one slide frame filled with 25 cysts into the soil. Each
treatment was replicated eight times in two independent experiments.
Nematode penetration was determined 16 days after inoculation. Potato
roots were rinsed with tap water to remove soil, blotted
on tissue
paper to remove excess water, and boiled in 0.1% lactic
acid fuchsin
(
7). After homogenization in an Ultra-Turrax (IKA-Werk),
the
juveniles were counted under a
stereomicroscope.
Data were analyzed for significance by analysis of variance and
Duncan's multiple-range test, using the software Statgraphics
Plus.
| |
RESULTS |
Analysis of EPS and LPS.
Analysis of LPS patterns by SDS-PAGE
confirmed the presence of LPS constructs in the LPS extract (Fig.
1). The LPS of R. etli G12
separated into five to six bands, the lowest of which represented truncated LPS consisting of lipid A-core region; the upper bands represented complete LPS molecules with O antigens of different sizes.
The LPS clearly possessed characteristic patterns of
Rhizobium spp. (4, 24). In the EPS extract of
Rhizobium etli G12, typical LPS bands were not detected.
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FIG. 1.
Pattern of EPS and LPS extracts from R. etli
G12 after SDS-PAGE and silver staining. Each lane was loaded with a
7.5-µl aliquot of the sample (10 mg ml1).
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|
The test for KDO verified the presence of the core region in the LPS of
R. etli G12 at concentrations of 15 nmol of KDO mg
of
LPS
1. The lack of KDO in the EPS extract excluded any
contamination
by LPS. The protein contents of the EPS and LPS extracts
were
low, reaching 0.25 and 0.38%, respectively. These data
demonstrated
that the silver-staining method had specifically stained
polysaccharide
bands and not
proteins.
Bioassays.
Our experiments confirmed the induction of systemic
resistance in potato roots against nematode infection by pretreatment with R. etli G12 (Fig. 2 and
4). The EPS of R. etli G12 showed no effect on G. pallida infection when applied as a root dip (Fig. 2). Treatment
with the LPS extract (1 mg ml1), however, resulted in a
significant (up to 44%) decrease in G. pallida infection of
potato roots (Fig. 3). Even the lowest LPS concentration (0.1 mg ml1) reduced nematode infection
by 40%.
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FIG. 2.
Penetration of G. pallida into potato roots
pretreated with Ringer solution (control), R. etli G12, or
its EPS, measured 16 days after nematode inoculation. Bars with
different letters are significantly different at P  0.05 (Duncan's multiple range test, n = 9).
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FIG. 3.
Penetration of G. pallida into potato roots
pretreated with Ringer solution (control), R. etli G12, or
different concentrations of its LPS, measured 16 days after nematode
inoculation. Bars with different letters are significantly different at
P 0.01 (Duncan's multiple range test, n = 9).
|
|
To clarify whether the LPS of
R. etli G12 act in potato
roots as an inducer of systemic resistance to
G. pallida,
bacterial
LPS and the parasite were applied spatially separated in a
split-root
system to prevent direct contact. Application of LPS (5 mg
per
pot) to one half of the split-root system caused a significant
(37%) systemic reduction in nematode penetration in the other
half of
the split-root system (Fig.
4). Living
cells of
R. etli G12 applied in the same manner reduced
nematode infection similarly,
by 34%. The results indicate that
surface components of
R. etli G12 act in potato roots as an
inducing factor of systemic resistance
to
G. pallida
infection.
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FIG. 4.
Penetration of G. pallida into the untreated
half of potato roots growing in the split-root system, 16 days after
the other root half was treated with Ringer solution (control), living
bacteria (R. etli G12), or bacterial LPS. Bars with
different letters are significantly different at P 0.05 (Duncan's multiple range test, n = 8).
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| |
DISCUSSION |
Previous studies demonstrated that specific rhizobacteria reduce
plant infection by various parasitic nematodes (21, 23, 32).
Recently, it was shown that the rhizobacterium R. etli G12
impaired infection by the potato cyst nematode G. pallida indirectly by inducing systemic resistance (9). Since the
plant defense capacity in potato roots was enhanced by both living and heat-killed cells of this strain, it was concluded that heat-stable surface structures such as EPS and/or LPS act as inducing agents.
In this study, we demonstrated that the EPS of R. etli G12
did not affect plant defense reactions in potato roots to nematode infection. Also, the EPS of another Rhizobium strain did not
affect the interaction with legumes (29). These authors
demonstrated that EPS-negative mutants induced levels of nodulation
similar to those induced by the wild-type strain. However, treatment of alfalfa with an EPS-negative mutant of Rhizobium meliloti
resulted in an accumulation of phenolics and callose (22).
It was suggested, therefore, that EPS function as a suppressor of plant
defense reactions, enabling the bacterium to infect the plant and grow endophytically. Since R. etli G12-mediated induced
resistance is also not associated with typical plant defense reactions
such as enhanced activity of pathogenesis-related proteins or increased lignin content, even though the bacteria colonize the roots locally (30), EPS of R. etli G12 may also act as a
suppressor of specific plant defense reactions. However, a role of EPS
as an inducing factor leading to a systemic reduction in nematode
infection throughout the root system can be ruled out.
We demonstrated that root dipping in the LPS solution of R. etli G12 reduced nematode infection significantly at
concentrations as low as 1 and 0.1 mg ml1. Furthermore, a
split-root experiment confirmed that the bacterium-free LPS of R. etli G12 were in large part responsible for systemic induced
resistance of potato to G. pallida infestation. Also, during
colonization of potato roots by R. etli G12, the bacterial LPS may be the decisive resistance inducer, since it is known from
studies of many gram-negative bacteria that LPS is released into the
environment during bacterial growth (31, 39). Inducer activity has also been shown for the LPS of the rhizobacterium P. fluorescens strain WCS417r towards
Fusarium wilt in carnation, radish, and
Arabidopsis (15, 36, 37). Similar to the results in our experiments (30), the induced resistance state was
not associated with enhanced production of pathogenesis-related
proteins (10, 25). Interestingly, LPS mutants of WCS417r
lacking the O-antigenic side chain of the LPS failed to protect radish
from Fusarium wilt even though they colonized the root to
the same extent as the wild-type strain (15). However, other
authors showed that in Arabidopsis the O-antigen-negative
mutant of WCS417r induced levels of protection similar to those induced
by the wild-type strain (37). To identify the LPS component
of R. etli G12 inducing systemic resistance of potato roots
to G. pallida attack, the potential roles of the O antigen,
core region, and lipid A of the rhizobial LPS are currently under investigation.
| |
ACKNOWLEDGMENTS |
We thank Petra Müller for advice on EPS and LPS analysis.
We thank the Deutsche Forschungsgemeinschaft for funding this project.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Pflanzenkrankheiten, Phytomedizin in Bodenökosystemen,
Universität Bonn, Nussalle 9, D-53115 Bonn, Germany. Phone: 49 228 732439. Fax: 49 228 732432. E-mail: rsikora{at}uni-bonn.de.
| |
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Applied and Environmental Microbiology, August 2000, p. 3515-3518, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
