Microbiology (2003), 149, 37–46
DOI 10.1099/mic.0.25859-0
Surface motility in Pseudomonas sp. DSS73 is
required for efficient biological containment of the
root-pathogenic microfungi Rhizoctonia solani and
Pythium ultimum
Jens B. Andersen,1 Birgit Koch,2 Tommy Harder Nielsen,2 Dan Sørensen,3
Michael Hansen,2 Ole Nybroe,2 Carsten Christophersen,3 Jan Sørensen,2
Søren Molin1 and Michael Givskov1
1
Section of Molecular Microbiology, Biocentrum-DTU, Building 301, Technical University of
Denmark, 2800 Lyngby, Denmark
Correspondence
Michael Givskov
2
[email protected]
Section of Genetics and Microbiology, Department of Ecology, The Royal Veterinary and
Agricultural University, Copenhagen, Denmark
3
Marine Chemistry Section, The H. C. Ørsted Institute, University of Copenhagen, Copenhagen,
Denmark
Received 8 July 2002
Revised
17 September 2002
Accepted 25 September 2002
Pseudomonas sp. DSS73 was isolated from the rhizoplane of sugar beet seedlings. This strain
exhibits antagonism towards the root-pathogenic microfungi Pythium ultimum and Rhizoctonia
solani. Production of the cyclic lipopeptide amphisin in combination with expression of flagella
enables the growing bacterial culture to move readily over the surface of laboratory media. Amphisin
is a new member of a group of dual-functioning compounds such as tensin, viscosin and
viscosinamid that display both biosurfactant and antifungal properties. The ability of DSS73 to
efficiently contain root-pathogenic microfungi is shown to arise from amphisin-dependent surface
translocation and growth by which the bacterium can lay siege to the fungi. The synergistic effects of
surface motility and synthesis of a battery of antifungal compounds efficiently contain and terminate
growth of the microfungi.
INTRODUCTION
Damping-off disease in sugar beet seedlings is caused by a
number of soil-borne plant pathogens, including the basidiomycete Rhizoctonia solani and the oomycete Pythium
ultimum. To protect crops against soil-borne diseases in
general, seeds are commonly treated with fungicides. Since
fungicides may affect human health and the environment
and since pathogens can develop resistance to fungicides,
bacterial inoculants exhibiting antagonism against plantpathogenic micro-organisms are receiving increased attention as environmentally friendly alternatives to the use of
chemical pesticides. One group of bacteria that shows
great promise with respect to protecting plant roots from
fungal-induced diseases is that containing the fluorescent
Pseudomonas spp. The biocontrol activity of these strains is
usually caused by the synthesis of one or more antifungal
factors, which include such diverse compounds as hydrogen
cyanide (Voisard et al., 1989), siderophores, pterines,
pyrroles (Shanahan et al., 1992), phenazines (Thomashow
& Weller, 1988), phloroglucinols (Shanahan et al., 1992),
peptides (Nielsen et al., 1999, 2000; Thrane et al., 2000),
proteases and chitinases (Nielsen et al., 1998; Dowling &
O’Gara, 1994). In fluorescent Pseudomonas strains, the
0002-5859 G 2003 SGM
biosynthesis of antifungal compounds is regulated by a
cascade of endogenous signals, which is channelled through
a sensor-kinase and response regulator encoded by gacAS
(Corbell & Loper, 1995; Gaffney et al., 1994; Laville et al.,
1992), sigma factors encoded by rpoS (Sarniguet et al., 1995)
and rpoD (Schnider et al., 1995), and quorum-sensing
systems (Pierson et al., 1998).
Among the great variety of antifungal metabolites produced
by fluorescent Pseudomonas spp., our attention has been
focused on the cyclic lipopeptides (Kochi et al., 1951;
Nielsen et al., 1998, 1999, 2000, 2002). In addition, there are
several examples of a close correlation between the
production of cyclic lipopeptides and the ability to carry
out surface motility on low-agar nutrient plates (Nielsen
et al., 2002). One well-studied phenomenon regarding
bacterial surface translocation and the involvement of
cyclic lipopeptides, such as serrawettins and rubiwettins, is
the swarming motility of Serratia spp. (Matsuyama et al.,
1990, 1992; Lindum et al., 1998). The swarming cells are
found almost exclusively in a motile layer present at the
perimeter of the expanding colony. This motile layer forms
a bacterial biofilm, which precedes the bulk of bacterial
mass. Serratia swarm cells are generally longer and more
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37
J. B. Andersen and others
flagellated than their counterparts propagated in liquid
media (the swimmer cells) and move within an encasement
of a self-produced ‘slime’ that surrounds the colony (see
review by Eberl et al., 1999). The morphology of swarm cells
combined with the production of biosurfactants as well as
the extraction of water from the underlying medium is
believed to reduce the surface friction which in turn
conditions the surface and as a consequence enables rapid
expansion of the growing bacterial culture (Matsuyama
et al., 1990, 1992; Bees et al., 2000). The surfactant
synthesized by Pseudomonas sp. DSS73 has been designated
amphisin. The complete structural elucidation of amphisin
has revealed that it is a cyclic lipopeptide consisting of an 11
amino acid cyclic peptide that is linked at the N-terminal
end to b-hydroxydecanoyl (Sørensen et al., 2001).
In the rhizosphere, the potential benefits of bacterial
motility include increased efficiency in nutrient acquisition,
avoidance of toxic substances, ability to translocate to
preferred hosts and access to optimal colonization sites
within them (Turnbull et al., 2001). Since root-pathogenic
microfungi appear to be highly motile in the rhizosphere, we
speculated that the surface motility of root-associated
biocontrol bacteria might play an important role in the
effective protection of plant roots against microfungal
attack. In this study, we characterized the surface motility
phenomenon exhibited by the Pseudomonas biocontrol
strain DSS73. Here, we present in vitro data implicating
surface motility as an important factor for ensuring efficient
impediment of the spreading mycelium of two plantpathogenic microfungi that are known to induce dampingoff disease in sugar beet seedlings.
METHODS
Strains, plasmids and growth conditions. The bacterial strains
and plasmids used in this study are listed in Table 1. The rootpathogenic microfungi employed as test fungi in biocontrol assays
were the basidiomycete R. solani (strain 92009; Danisco Seed,
Holeby, Denmark) and the oomycete Pythium ultimum (strain
92001; Danisco Seed) (Wolffhechel et al., 1992), both capable of
inducing damping-off disease in sugar beet. Both fungi were maintained as described by Nielsen et al. (1998). The basic growth
medium used was ABT minimal medium [AB minimal medium
(Clark & Maaløe, 1967) containing 2?5 mg thiamin l21] supplemented with different carbon sources (see below). When appropriate,
the medium was supplemented with the following amounts of antibiotics: 100 mg ampicillin ml21, 25 mg kanamycin ml21 and 10 mg
tetracycline ml21 for Pseudomonas strains, and 25 mg kanamycin
ml21 for Serratia liquefaciens.
Sugar beet seed extract. This was prepared by incubating 50 g
granulated sugar beet seeds (cv. ‘Madison’; Danisco Seed) with
200 ml ABT minimal medium on a horizontal shaker operating at
200 r.p.m. for 3 h at 37 ˚C. Next, the extract was successively filtered
through cloth, Whatman paper no. 1 (Millipore), Whatman paper
no. 3 (Millipore), a 0?8 mm pore size syringe filter (Sartorius) and a
0?2 mm pore size syringe filter (Sartorius AG) to produce a sterile
sugar beet seed extract.
Surface motility assays. These were carried out on ABT minimal
medium plates supplemented with 0?4 % (w/w) glucose and 0?4 %
(w/w) Casamino acids (Difco) and solidified with 0?6 % Bacto Agar
(Difco). In the following, these are referred to as the standard surface motility plates. When appropriate, the medium was supplemented with other carbon sources (see below) and different agar
concentrations. The media were poured as 25 ml aliquots into 9 cm
sterile Petri dishes and the resulting standard surface motility plates
were dried for 2 h at 20 ˚C prior to inoculation with 1 ml culture
droplets (harvested and washed twice with 1 ml of 0?9 % NaCl) of
wild-type DSS73, DSS73-15C2 (AmsY2), DSS73-12H8 (GacS2) or
DSS73-12H8 (GacS2) carrying the plasmid pEMH97 (this plasmid
carries the gacS gene of Pseudomonas syringae pv. syringae).
Following 20 h incubation at 20 ˚C, all surface motility plates were
visually inspected and the surface motility phenotypes of the respective strains were recorded. All assays were done in triplicate and
images of the representative surface motility phenotypes were captured using a Kodak DC240 digital camera. The resulting images
were processed using Photoshop 5.5. To obtain bacterial inocula,
strains were cultivated in liquid ABT medium containing 0?4 % glucose (w/w) and 0?4 % Casamino acids (w/w) at 20 ˚C for 24 h.
Subsequently, 1 ml aliquots were harvested by centrifugation (2 min
at 10 000 g) and washed twice with 1 ml of 0?9 % NaCl.
Table 1. Bacterial strains and plasmids used in this study
Strain/plasmid
Strain
DSS73 Pseudomonas sp.
DSS73-15C2 Pseudomonas sp.
(AmsY2)
DSS73-12H8 Pseudomonas sp.
(GacS2)
S. liquefaciens MG1
S. liquefaciens PL10
Plasmid
pLAFR3
pEMH97
38
Relevant genotype/characteristics
Wild-type; amphisin producer
amsY mutant derived from DSS73;
amphisin-deficient; Kmr
gacS mutant derived from DSS73;
amphisin-deficient; Kmr
Wild-type; serrawettin W2 producer
swrA swrI double mutant derived from MG1;
serrawettin W2-deficient; Kmr, Strepr
Cloning vector; Tcr
pLAFR3 encoding gacS isolated from
Pseudomonas syringae pv. syringae; Tcr
Reference
Nielsen et al. (2002)
Koch et al. (2002)
Koch et al. (2002)
Givskov et al. (1988)
Lindum et al. (1998)
Staskawicz et al. (1987)
Hrabak & Willis (1992)
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Microbiology 149
Surface motility studies of Pseudomonas sp. DSS73
Surfactant complementation assays. Culture droplets (1 ml)
were inoculated onto standard surface motility plates (see above)
supplemented with 0, 1?25, 2?5, 5 or 10 mg ml21 of amphisin (a
cyclic lipopeptide isolated from Pseudomonas sp. DSS73) (Sørensen
et al., 2001; Nielsen et al., 2002), tensin (a cyclic lipopeptide isolated
from Pseudomonas fluorescens 96.578) (Nielsen et al., 2000), viscosinamid (a cyclic lipopeptide isolated from Pseudomonas fluorescens
DR54) (Nielsen et al., 1999), serrawettin W2 (a cyclic lipopeptide
isolated from S. liquefaciens MG1 (Lindum et al., 1998), NP40
(Nonidet P40, an artificial surfactant/non-ionic detergent; Sigma) or
Triton X-100 (a detergent; Sigma). Following 24 h incubation at
20 ˚C, all plates were visually inspected and the surface motility
phenotypes of the respective strains were recorded. All complementation assays were repeated three times and digital images of the
representative surface motility phenotypes of the respective strains
were captured and processed as described in the ‘Surface motility
assays’ section of Methods.
Nutritional requirements for surface motility. Aliquots (25 ml)
of ABT minimal medium containing 0?6 % Bacto agar and either
0?4 % (w/w) glucose and 0?4 % (w/w) Casamino acids, 0?4 % (w/w)
Casamino acids, 0?4 % (w/w) glucose, 0?4 % (w/w) D-xylose or 50 %
sugar beet extract were poured into sterile Petri dishes. The plates
were dried for 2 h at 20 ˚C and then inoculated with 1 ml culture
droplets. Following 20 h incubation at 20 ˚C, all plates were visually
inspected for surface motility. Since all of the strains employed in
the experiment grew slowly on plates containing D-xylose as carbon
source, their ability to perform surface motility on D-xylose plates
was re-evaluated following 7 days incubation at 20 ˚C. All strains
were tested three times for their ability to perform surface motility
on the plates described above; based on these triplicates a rough estimate of the maximal colony expansion velocity was obtained by
measuring the increase in colony radius as a function of time.
Biocontrol assays. To compare the abilities of strains DSS73,
DSS73-15C2 (AmsY2), DSS73-12H8 (GacS2) and DSS73-12H8
(GacS2) carrying pEMH97 to limit the growth of the root-pathogenic
microfungi R. solani and Pythium ultimum, the relevant bacterial
and fungal strains were co-cultivated on standard surface motility
plates and on nutrient agar plates composed of ABT minimal
medium containing 0?4 % (w/w) glucose, 0?4 % (w/w) Casamino
acids and 2 % Bacto agar (Difco). A 1 ml pre-culture of each bacterial strain was inoculated 2?5 cm away from the centre of a small
agar plug containing mycelium of either R. solani or Pythium ultimum, and the plates were incubated at 20 ˚C. As a control, agar
plugs containing mycelium of R. solani and Pythium ultimum were
also inoculated alone onto the same types of nutrient plates.
Following 24 and 48 h co-cultivation, all plates were visually
inspected and the ability of each bacterial strain to inhibit mycelium
spreading was evaluated. All biocontrol assays and appropriate controls were done in triplicate and digital images of the representative
biocontrol phenotypes were captured and processed as described in
the ‘Surface motility assays’ section of Methods. To address the viability of the microfungi contained by strain DSS73 on standard surface motility plates (see Fig. 4A as an example) agar plugs were
withdrawn from the outer edge of the mycelium and from the area
close to the original fungal inoculation site. These plugs were transferred to nutrient plates composed of ABT minimal medium containing 0?4 % (w/w) glucose, 0?4 % (w/w) Casamino acids, 2 %
Bacto agar and 10 mg tetracycline ml21 (tetracycline was added to
prevent the growth of biocontrol strain DSS73). As positive controls,
agar plugs containing non-challenged mycelium of R. solani and
Pythium ultimum were inoculated onto the same types of nutrient
plates. Following 3 days incubation at 20 ˚C, the plates were visually
inspected for growth of the respective fungus. All viability assays
were repeated three times.
Electron microscopy. Bacteria from the centre and the leading
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edge of a moving colony were analysed with respect to their cell
morphology and presence of flagella using electron microscopy.
Bacteria from the centre or the margin of three different surface
motile colonies of strain DSS73 were gently scraped off with a toothpick and subsequently suspended in a drop of water mounted on
top of a carbon-coated copper grid (mesh: 200) (Electron Microscopy Sciences). Next, the six grids were washed once with distilled
water and stained with 1 % uranyl acetate. The negatively stained
cells were visualized with a transmission electron microscope
(Phillips TEM, CM 100) equipped with a SIS Megaview II digital
camera system; the resulting electron micrographs were processed
using the ANALYSIS software package (SIS). From the resulting electron micrographs, mean cell size and mean number of flagella were
estimated.
RESULTS
Surface motility of strain DSS73
In a recent screening for novel sugar beet protective strains,
Pseudomonas sp. DSS73 was isolated from the rhizoplane of
sugar beet seedlings as a biosurfactant-producing strain that
exhibited antagonism towards the root-pathogenic microfungi Pythium ultimum and R. solani (Nielsen et al., 2002).
This study revealed that the ability to synthesize biosurfactants is a quite common property in root-associated,
fluorescent Pseudomonas spp. Furthermore, many of the
strains were found to form expanding colonies on semisolid laboratory media. This suggests a connection between
biosurfactant production and the ability to carry out surface
motility (Nielsen et al., 2002). We decided to investigate
conditions affecting the surface motility of strain DSS73.
Within the first 12–16 h of incubation, DSS73 formed a
morphologically normal colony with a diameter of approximately 2–4 mm on standard surface motility plates.
However, as cell density increased further, small peninsulas
of cells were eventually generated at the colony perimeter
and were observed to move vertically away from the colony.
These peninsulas never increased to more than 3 mm in
length and 1 mm in width, as the free space between
neighbouring peninsulas was continuously filled in by
residual growth. As a result, the expanding colony exhibited
small ‘tongue’-like structures of biofilms at its perimeter.
Once this phenomenon had been triggered, the diameter of
the DSS73 colony was observed to expand by 8 mm h21,
rendering the strain capable of covering the entire surface of
a 9 cm Petri dish within 24 h after inoculation (Fig. 1).
Microscopic inspection revealed that cells at the perimeter
of the colony formed a highly motile swarm, and neither
single cells nor small groups of cells were ever observed to
escape/leave the dense colony. This motility pattern is
very similar to the swarming motility observed with
S. liquefaciens (Eberl et al., 1996, 1999). Since the hallmark
of true swarming motility is considered to involve the
differentiation of cells into an elongated, multinucleated and
profusely flagellated form (Harshey, 1994), we decided to
clarify if surface contact induced differentiation in strain
DSS73. Electron microscopy revealed that bacteria isolated
from the edge of the expanding colony were on average
approximately 2 mm long and possessed four to six polar
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39
J. B. Andersen and others
Fig. 1. Surface motility phenotypes of Pseudomonas sp. DSS73 and mutant derivatives. (A) DSS73, (B) DSS73-15C2
(AmsY2), (C) DSS73-12H8 (GacS2) and (D) DSS73-12H8 (GacS2) carrying pEMH97 were cultivated on ABT/glucose/
Casamino acids plates solidified with 0?6 % agar (standard surface motility plates). (E) DSS73-15C2 (AmsY2) and (F)
DSS73-12H8 (GacS2) were cultivated on ABT/glucose/Casamino acids plates containing 2?5 mg amphisin (ml medium)21
and solidified with 0?6 % agar. All plates were inoculated with 1 ml of an overnight culture of the respective strain; digital
images were captured following 20 h incubation at 20 ˚C.
flagella, whereas cells from the centre of the colony were
approximately 1 mm long and possessed just one polar
flagellum (Fig. 2). We therefore decided to refer to the
phenomenon as surface motility rather than swarming
motility.
Propagation of strain DSS73 on a series of nutrient plates
with increasing agar contents revealed that the expansion
velocity declined when the agar content exceeded 0?7 %. At
1 % agar, the expansion velocity of the colony was drastically
reduced (data not shown). In the presence of 1?2 % agar,
surface motility was completely abolished. This gave rise to
colonies that were morphologically indistinguishable from
colonies propagated on nutrient plates containing 1?5 and
2 % agar (data not shown). In comparison, Proteus mirabilis
(Allison & Hughes, 1991) and Vibrio parahaemolyticus
(McCarter & Silverman, 1990) have been demonstrated to
swarm at agar concentrations up to 2 %.
Amphisin production is required for surface
motility of strain DSS73
The amphisin synthesized by strain DSS73 might promote
or enable surface translocation. In a recent study by Koch
et al. (2002), two surfactant-negative mutants, DSS73-15C2
40
(AmsY2) and DSS73-12H8 (GacS2), carrying mutations
in amsY (encoding the putative amphisin synthetase) and
gacS, respectively, were isolated. These two mutants were
subsequently demonstrated to be defective in the synthesis
of amphisin. When stab inoculated on nutrient plates
solidified with 0?3 % agar, the swimming motility phenotypes of DSS73-15C2 (AmsY2) and DSS73-12H8 (GacS2)
were indistinguishable from the swimming motility phenotype exhibited by the parent wild-type strain, DSS73 (data
not shown). This indicates the presence of functional flagella
and a fully operational chemotaxis apparatus in both of the
amphisin-deficient mutants. DSS73-15C2 (AmsY2) and
DSS73-12H8 (GacS2) failed to carry out surface motility on
0?6 % agar plates unless the medium was supplemented
with 2?5 mg amphisin ml21 (Fig. 1B, C, E and F). This
clearly demonstrated that amphisin is the surface-tensionreducing compound that enables strain DSS73 to move
over the agar surface (Fig. 1A). In addition, surface motility
could be restored in the gacS mutant upon introduction of
pEMH97 encoding the heterologous wild-type gacS gene
from Pseudomonas syringae pv. syringae (Hrabak & Willis,
1992), supporting the finding of Koch et al. (2002) that
amphisin synthesis is regulated by gacS (compare Fig.1C
and D).
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Microbiology 149
Surface motility studies of Pseudomonas sp. DSS73
Table 2. Surface motility in the presence of different surfactants
Surfactant*
Surface motility of strain3
DSS73 DSS73-15C2 DSS73-12H8 MG1 PL10
(AmsY2)
(GacS2)
None
Amphisin4
Tensin4
Viscosinamid4
Serrawettin W24
NP401
Triton X-1001
+
+
+
+
+
+
+
2
+
+
+
+
2
2
2
+
+
+
+
2
2
+
+
+
+
+
+
+
2
+
+
+
+
+
+
*Surfactant added to ABT minimal medium supplemented with 0?4 %
glucose, 0?4 % Casamino acids and 0?6 % agar prior to casting.
3Surface motility was evaluated following 24 h incubation at 20 ˚C;
+, surface motility; 2, no surface motility.
4Final concentration of surfactant in the plates was 2?5 mg ml21.
1Final concentration of surfactant in the plates was 10 mg ml21.
Fig. 2. Morphology of Pseudomonas sp. DSS73 cells isolated
from a swarming colony cultivated on nutrient plates solidified
with 0?6 % agar. (A) Electron micrograph of DSS73 taken
directly from the centre of a swarming colony. At the centre,
cells were approximately 1 mm and were observed to express
just one polar flagellum. (B) Electron micrograph of DSS73
taken directly from the edge of a surface-motile colony.
2
Surface motility of the two mutants, DSS73-15C2 (AmsY )
and DSS73-12H8 (GacS2), was also restored by exogenous
tensin (2?5 mg ml21), viscosinamid (2?5 mg ml21) or serrawettin W (2?5 mg ml21) whereas the addition of synthetic
surfactants such as NP40 and Triton X-100 failed to
promote surface motility (Table 2). NP40 and Triton X-100
restored swarming motility in the surfactant-deficient
S. liquefaciens strain PL10 (Table 2).
Nutritional requirements of surface motility in
strain DSS73
Unlike most surface-motile bacteria, the surface motility of
strain DSS73 did not depend on the presence of Casamino
acids, peptone or any single amino acid in the growth
medium (Table 3). The only nutritional requirement of the
bacterium for it to carry out rapid surface motility was a
mineral medium containing an easily metabolizable carbon
source such as glucose. However, it is noteworthy that a
significantly higher colony expansion velocity was obtained
for strain DSS73 in the presence of Casamino acids (the sole
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carbon source). D-Xylose (a sugar compound often
encountered in root exudates) turned out to be a very
poor carbon source for strain DSS73, as it took 3–4 days for
the strain to form a normal-sized colony (Table 3).
Nevertheless, surface motility was eventually triggered at
day 4 of incubation, and within the next six days the colony
reached the edge of the plate. Instead of the usual confluent
spreading, strain DSS73 was observed to move in dendrites
when propagated on D-xylose plates. Whether this changed
colony morphology in DSS73 was the result of increased
surface tension due to dehydration of the plate during the
long incubation or was dictated by the D-xylose is not
known.
Recently it was reported that amphisin production on
glucose-supplemented minimal medium is increased sixfold
upon the addition of a sugar beet seed extract to the medium
(Koch et al., 2002). Interestingly, plates supplemented with
sugar beet seed extract as the sole carbon source enabled
strain DSS73 to traverse the standard surface motility plate
with a speed indistinguishable from that recorded on
Casamino acids (Table 3).
Antifungal properties of strain DSS73 are
regulated by GacS
The ability of strain DSS73 to inhibit the growth of the rootpathogenic microfungi Pythium ultimum and R. solani is
believed to originate from the production and excretion of
amphisin, chitinases, proteases and hydrogen cyanide by the
bacterium (Nielsen et al., 2002; Koch et al., 2002).
Interestingly, the production of these antifungal metabolites
requires a functional gacS gene whereas only the production
of amphisin seems to be impaired in the amsY mutant (Koch
et al., 2002). To characterize the antifungal properties of
DSS73 in further detail, we compared the abilities of strains
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J. B. Andersen and others
Table 3. Surface motility in the presence of different carbon sources
Carbon source*
None4
Glucose and Casamino acids4
Casamino acids4
Glucose4
D-Xylose1
Extract of sugar beet seeds4
Surface motility of strain3
DSS73
DSS73-15C2 (AmsY2)
DSS73-12H8 (GacS2)
MG1
PL10
No growth
+++
+++
++
+
+++
No growth
2
2
2
2
2
No growth
2
2
2
2
2
No growth
+++
+++
2
2
++
No growth
2
2
2
2
2
*Carbon source added to plates containing ABT minimal medium and 0?6 % agar.
32, Surface immobile; +, maximal colony expansion velocity ,2 mm h21; ++, maximal colony expansion velocity 2–4 mm h21; +++, maximal
colony expansion velocity .4 mm h21.
4Evaluation of surface motility was done following 20 h incubation at 20 ˚C.
1Due to slow growth of all strains tested, evaluation of surface motility was done following 7 days incubation at 20 ˚C.
DSS73, DSS73-15C2 (AmsY2), DSS73-12H8 (GacS2) and
DSS73-12H8 (GacS2) carrying pEMH97 to inhibit the
growth of R. solani and Pythium ultimum. Following cocultivation of strain DSS73 and R. solani on 2 % agar plates,
a mycelium-deficient zone developed around the bacterial
colony, confirming the antifungal properties of DSS73
(Fig. 3A). A similar result was obtained when the mycelium
of R. solani approached the bacterial colony of the amsY
mutant, DSS73-15C2 (AmsY2) (Fig. 3B). In striking contrast, R. solani appeared unaffected by the presence of
the gacS mutant DSS73-12H8 (GacS2) when co-cultivated
under identical conditions. Indeed, the mycelium of the
fungus was eventually seen to cover the colony formed by
DSS73-12H8 (GacS2), signifying a complete absence of
antifungal metabolites in this strain (Fig. 3C). However,
upon provision of the wild-type gacS gene product from
Pseudomonas syringae pv. syringae, present on pEMH97, to
DSS73-12H8 (GacS2) a mycelium-deficient zone was
maintained around the bacterial colony, demonstrating
that the antifungal phenotype had been restored (Fig. 3D).
As similar observations were made when Pythium ultimum
was challenged with strains DSS73 (AmsY2), DSS73-15C2
(GacS2), DSS73-12H8 and DSS73-12H8 (GacS2) carrying
pEMH97 (data not shown), these results clearly show that
synthesis of antifungal metabolites in DSS73 requires the
presence of a functional gacS gene. This strongly suggests
that expression of the antifungal properties of strain DSS73
is controlled by a gacS/gacA-encoded two-component
system similar to other Pseudomonas spp. (Chancey et al.,
1999; Laville et al., 1992; Gaffney et al., 1994; Pfender et al.,
1994; Corbell & Loper, 1995; Schmidli-Sacherer et al., 1997).
Surface motility promotes containment of
microfungi
Although strain DSS73 clearly exhibited antagonism
towards R. solani and Pythium ultimum on 2 % agar nutrient plates (Fig. 3A, and data not shown), both microfungi
eventually ended up occupying as much as 98 % of the
available surface. We envisaged that this outcome could be
reversed if both organisms were propagated on a medium
that sustained bacterial surface motility. To confirm this, we
evaluated the abilities of strains DSS73, DSS73-15C2
(AmsY2) and DSS73-12H8 (GacS2) to impede mycelium
Fig. 3. Antifungal properties of Pseudomonas sp. DSS73 and mutant derivatives towards the root-pathogenic microfungus
R. solani on 2 % agar plates. R. solani was challenged with (A) DSS73, (B) DSS73-15C2 (AmsY2), (C) DSS73-12H8
(GacS2) or (D) DSS73-12H8 (GacS2) carrying pEMH97 on ABT/glucose/Casamino acids plates solidified with 2 % agar. All
images were captured following 48 h incubation at 20 ˚C.
42
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Microbiology 149
Surface motility studies of Pseudomonas sp. DSS73
spreading of both R. solani and Pythium ultimum on
standard surface motility plates. As expected, the antifungal
properties of the non-surface-motile strains DSS73-15C2
(AmsY2) and DSS73-12H8 (GacS2) did not change
(compare Fig. 3B with Fig. 4B and Fig. 3C with Fig. 4C).
R. solani readily colonized 98 and 100 %, respectively, of
the nutrient plate surface within 60 h of co-cultivation. In
striking contrast, mycelium spreading of R. solani was
severely hampered when it was co-cultivated with the
surface-motile strain DSS73 (Fig. 4A). Once the DSS73
colony started to expand, contact between the preceding
bacterial biofilm and the fungus was achieved within a few
hours. Microscopic inspection revealed that upon contact,
the biofilm was partly dissolved as planktonic bacteria
moved along the hyphae by swimming motility while the
areas between the hyphae were filled in by the biofilm swarm
(not shown). In this way DSS73 rapidly traversed the fungal
mycelium and the fungus was completely surrounded by the
expanding bacterial colony. Apparently, due to the spatial
limitation, the fungus seemed to be unable to expand.
Furthermore, the trapped R. solani was no longer able to
maintain mycelium growth (Fig. 4A). The activity of strain
DSS73 seemed to be lethal to the fungus, as the fungus failed
to resume mycelium growth when transferred to fresh
nutrient plates (data not shown). Supporting the assumption that GacS/GacA is a key regulator of biocontrol in
DSS73, almost identical observations were made when
R. solani was challenged with strain DSS73-12H8 (GacS2)
carrying pEMH97 on standard surface motility plates. In
accordance with the restoration of both surface motility
and synthesis of antifungal metabolites, the presence of a
functional gacS product in trans clearly enabled the strain to
impede mycelium spreading of the fungus (Fig. 4D).
Cultivation of R. solani on standard surface motility plates in
which the growth medium had been supplemented with
varying amounts of amphisin revealed that fungal growth
was not significantly affected until a concentration of
approximately 10 mg amphisin (ml medium)21 was reached
(data not shown). In contrast, 2?5 mg amphisin (ml
medium)21 was the minimum concentration required to
fully restore surface motility of the amsY and gacS mutants
(see Table 3). Therefore, it was possible to clarify whether
spatial limitations alone or in combination with synthesis
of antifungal agents were responsible for the observed
efficient containment of the fungus. To address this
question, R. solani was challenged with DSS73-12H8
(GacS2) and DSS73-15C2 (AmsY2), respectively, on
standard surface motility plates supplemented with 2?5 mg
amphisin ml21. Under these conditions, the ability of the
amsY mutant to block mycelium spreading was restored
(compare Fig. 4B and E). Once surrounded by the bacterial
Fig. 4. Antifungal properties of Pseudomonas sp. DSS73 and mutant derivatives towards the root-pathogenic microfungus
R. solani on standard surface motility plates. R. solani was challenged with (A) DSS73, (B) DSS73-15C2 (AmsY2), (C)
DSS73-12H8 (GacS2) or (D) DSS73-12H8 (GacS2) carrying pEMH97 on ABT/glucose/Casamino acids plates solidified
with 0?6 % agar. (E, F) The fungus was challenged with either (E) DSS73-15C2 (AmsY2) or (F) DSS73-12H8 (GacS2) on
ABT/glucose/Casamino acids plates containing 2?5 mg amphisin (ml medium)21 and solidified with 0?6 % agar. All images
were captured following 64 h incubation at 20 ˚C.
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43
J. B. Andersen and others
colony, R. solani was never observed to resume growth, not
even following prolonged incubation. A somewhat different
scenario emerged following co-cultivation of R. solani
with the gacS mutant on standard surface motility plates
supplemented with 2?5 mg amphisin ml21. Although fully
capable of moving over the surface of the agar, strain
DSS73-12H8 (GacS2) completely failed to stop the
spreading mycelium. Even when completely surrounded
by the bacterial colony, R. solani was still observed to
maintain mycelium propagation into the agar beneath the
DSS73-12H8 (GacS2) culture. Similar results were obtained
by replacing R. solani with Pythium ultimum in all of the
experiments described in this section (data not shown). This
strongly suggests that the fungus had been effectively
contained by strain DSS73 as a result of a combination of
spatial limitations and a cocktail of antifungal agents.
DISCUSSION
The fluorescent Pseudomonas sp. DSS73 was originally
isolated from the rhizoplane of sugar beet seedlings as a
biosurfactant-producing strain capable of inhibiting the
growth of the root-pathogenic microfungi R. solani and
Pythium ultimum (Sørensen et al., 2001; Nielsen et al., 2002).
This strain displayed a convincing surface motility phenotype when propagated on nutrient plates containing
0?4–1 % agar. A study by Nielsen et al. (2002) revealed
that the ability to produce biosurfactants is a quite common
phenotype in root-associated fluorescent Pseudomonas spp.,
suggesting that surface motility in vivo requires the production of biosurfactants. At the macroscopic level, strain
DSS73 predominantly gave rise to a concentric, confluent,
moving colony that was distinctly different from the
dendrite colony pattern that arose during surface translocation of Pseudomonas aeruginosa (Rashid & Kornberg, 2000;
Köhler et al., 2000) and Pseudomonas syringae pv. syringae
(Kinscherf & Willis, 1999). With strain DSS73, cells located
at the edge of the expanding colony moved rapidly in swirls
that closely resembled the swarming pattern reported for
cells at the rim of swarming colonies of S. liquefaciens (Eberl
et al., 1996, 1999). Since planktonic cells were never
observed, the surface motility of strain DSS73 is likely to
originate from the multicellular behaviour of the cells
present in the motile biofilm part of the expanding colony.
However, surface translocation of strain DSS73 was clearly
not accompanied by any substantial increase in flagellation
and cell elongation, and was therefore almost indistinguishable from surface-motile colonies of Pseudomonas aeruginosa (Rashid & Kornberg, 2000; Köhler et al., 2000). Our
genetic analysis indicated that the surface motility of strain
DSS73 occurs within the framework of the GacS/GacAcontrolled regulon, as has been reported for Pseudomonas
syringae (Kinscherf & Willis, 1999), and is not controlled by
quorum sensing, as reported for S. liquefaciens (Eberl et al.,
1996; Lindum et al., 1998), Pseudomonas aeruginosa (Köhler
et al., 2000) and Burkholderia cepacia (Huber et al., 2001).
Strain DSS73 failed to activate any of our N-acyl-Lhomoserine lactone (AHL) sensors, which covered the
44
entire range of known AHL signals (data not shown). In fact,
during the screening for and characterization of sugar-beetroot-associated, biosurfactant-producing Pseudomonas
spp., only three out of 80 surfactant-producing isolates
appeared capable of synthesizing AHL signal molecules.
Similar to Serratia spp., the ability of Pseudomonas sp.
DSS73 to move over the agar surface was clearly attributed
to its production of an extracellular biosurfactant, since the
presence of purified amphisin in the growth medium
restored this phenotype to the amsY and gacS mutants. In
contrast to S. liquefaciens, which is highly promiscuous with
respect to accepting surfactants for swarming motility
(Lindum et al., 1998; Eberl et al., 1999), amphisin and other
closely related cyclic lipopeptides are prerequisites for the
surface motility of strain DSS73. This suggests that not only
surface tension reduction but also additional physical/
chemical properties of these closely related cyclic biosurfactants are crucial for the surface motility of DSS73.
Fungal growth was inhibited on agar plates supplemented
with purified amphisin at a minimum concentration of
10 mg ml21, which is four times the concentration required
to efficiently promote bacterial surface motility. Therefore,
amphisin can be considered a dual-functioning compound.
For fungal antagonism, however, DSS73 relies not on
amphisin alone. It produces a battery of antagonistic agents
such as proteases, chitinases and hydrogen cyanide, all of
which are controlled by the GacS/GacA system (Koch et al.,
2002). Our results clearly demonstrate that the efficient
elimination of competing fungi is due to the synergistic
effect of surface motility and synthesis of antifungal agents.
Without these factors the bacteria were unable to completely
contain the fungi.
Our data also suggest that surfactant activity is a more
important property than the antimicrobial action of
amphisin. However, the artificial nature of the experimental
set-up should be kept in mind. If the hardness of the surface
in vivo prevents the bacterial culture moving over the
surface, the antimicrobial activity of amphisin may be of
greater value to the producer. With a MIC of 10 mg ml21,
amphisin clearly retains its potency as an antimicrobial
compound.
Expression of the total antimicrobial cocktail (including
motility) produced by fluorescent Pseudomonas spp. is
controlled by the GacS/GacA system. Therefore, only a
single signal is required to mount the entire protective
arsenal of the bacteria. In a recent study, it was demonstrated
that synthesis of amphisin was increased approximately
sixfold when strain DSS73 was cultivated in the presence of a
sugar beet seed extract (Koch et al., 2002). Since the growth
rate of the organism was unaffected, this result indicates the
presence of a signal compound in the seed extract. The
structure of this putative signal compound has not yet been
elucidated; however, experiments have revealed that signal
transduction leading to increased transcription of amsY
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Microbiology 149
Surface motility studies of Pseudomonas sp. DSS73
clearly requires a functional gacS/gacA system (Koch et al.,
2002).
The apparent interplay between strain DSS73 and the sugar
beet seed might be considered as being mutually beneficial.
Initially, the sugar beet seed supplies the bacteria with
nutrients and releases signals that induce production of
amphisin. We speculate that this enables a culture of DSS73
to efficiently expand on the seed surface, but in addition it
also recruits the entire protective arsenal of the bacterium.
In the environment, multicellular behaviour is considered
an advantage for bacteria. Previous work by Eberl et al.
(1997) and Mallory et al. (1983) strongly suggests that the
formation of flocks offers bacteria protection against grazing
protozoa. Similarly, in the cause of infection, Pseudomonas
aeruginosa grows in biofilms that protect it from the action
of leukocytes (Høiby et al., 2001; Costerton et al., 1999). In
this latter case, control over surface motility, biofilm
architecture and the formation of a cocktail of virulence
factors is integrated by quorum sensing. It is tempting to
speculate that biocontrol bacteria, which coordinate their
effort and work in a flock, are more successful in their
interactions with other microbes and higher eukaryotes than
their planktonic counterparts.
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ACKNOWLEDGEMENTS
This work was supported by the Danish Agricultural and Veterinary
Research Council (J. no. 9702796). We thank Linda Stabell and Anne
Kirstine Nielsen for their excellent technical assistance and David K.
Willis (Department of Plant Pathology, University of Wisconsin,
Madison, WI 53706, USA) for providing plasmid pEMH97.
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