Environmental Microbiology (2010) 12(6), 1391–1398
doi:10.1111/j.1462-2920.2009.02022.x
Fungal mycelia allow chemotactic dispersal of
polycyclic aromatic hydrocarbon-degrading bacteria
in water-unsaturated systems
emi_2022
Shoko Furuno,1 Katrin Päzolt,2 Cornelia Rabe,1
Thomas R. Neu,2 Hauke Harms1 and Lukas Y. Wick1*
1
Helmholtz Centre for Environmental Research – UFZ,
Department of Environmental Microbiology, 04318
Leipzig, Germany.
2
Helmholtz Centre for Environmental Research – UFZ,
Department of River Ecology, 39114 Magdeburg,
Germany.
Summary
Contaminant biodegradation in soil is frequently
limited by hindered physical access of bacteria to
the contaminants. In the frame of the development
of novel bioremediation approaches based on ecological principles, we tested the hypothesis that
fungal networks facilitate the movement of bacteria
by providing continuous liquid films in which gradients of chemoattractants can form and chemotactic
swimming can take place. Unlike bacteria, filamentous fungi spread with ease in water-unsaturated
soil. In a simple laboratory model of a waterunsaturated environment, we studied the movement
of polycyclic aromatic hydrocarbon-degrading
Pseudomonas putida PpG7 (NAH7) along a mycelium of Pythium ultimum. Some undirected dispersal
was observed in the absence of a chemoattractant
or when the non-chemotactic derivative strain
P. putida G7.C1 (pHG100) was used. The bacterial
movement became fourfold more effective and
clearly directed when the chemotactic wild type was
used and salicylate was present as a chemoattractant. No dispersal of bacteria was found in the
absence of the fungus. These findings point at a role
of mycelia for the translocation of chemicals and
microorganisms. The results suggest that fungi
improve the accessibility of contaminants in waterunsaturated environments.
Received 28 May, 2009; accepted 25 June, 2009. *For correspondence. E-mail [email protected]; Tel. (+49) 341 2351316;
Fax (+49) 341 2351351.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd
1391..1398
Introduction
The capacity of microorganisms to rapidly sense and
adapt to environmental changes is an important factor for
the stability of microbial ecosystem services, such as
contaminant biodegradation in soil (de Lorenzo, 2008;
Miller et al., 2009). Marx and Aitken (2000), for instance,
have shown that bacterial chemotaxis, i.e. the ability to
sense and move along chemical concentration gradients,
enhances the biodegradation of naphthalene, a polycyclic
aromatic hydrocarbon (PAH), in bioavailability-limited
heterogeneous systems (for a review: Harms and Wick,
2006; Miller et al., 2009). Bacterial motility and chemotaxis in porous media, however, are still a field full of
unknowns and experimental evidence for its importance is
limited (Ford and Harvey, 2007). The efficiency of bacterial chemotaxis in bioremediation strongly depends on the
effective mobility of bacteria within the system. As bacteria require high soil matric potentials or at least continuous
liquid films for swarming and swimming (Or et al., 2007),
directed chemotactic dispersal is restricted in waterunsaturated environments. This may affect the bioaccessibility of patchy, hydrophobic organic compounds
(Semple et al., 2007), as average distances between bacterial microcolonies in soil are supposed to be in the range
of 10-4 m (Bosma et al., 1997). Microbial dispersal and
substrate mobilization are hence needed to overcome the
distance between substrates and organisms. The strategy
of filamentous fungi is to enlarge their external surface
and to develop mycelia of high fractal dimension that
optimally exploit the three-dimensional space containing
the substrate (Nakagaki et al., 2004). Contrary to bacteria, the habitat of fungi is not restricted to water films.
Fungal hyphae easily breach through air–water interfaces
and form dense networks of up to 20 000 km length per
cubic meter of soil (Pennisi, 2004). Importantly, by doing
so they connect saturated and unsaturated soil pores
(Wessels, 1997). Several reports on the role of fungal
mycelia on ‘underground networking’ for nutrient translocation and provision to bacteria in the hyphosphere
(Bending et al., 2006) and shaping of communities above
and below the earth’s surface (Whitfield, 2007) have been
published. As both bacteria and fungi are important
degraders of (anthropogenic) organic substances in soil,
1392 S. Furuno et al.
fundamental knowledge of bacteria–fungus interactions is
also essential for the development of novel bioremediation approaches based on ecological principles. For
instance, it has been shown that liquid films developing
around hydrophilic fungi can be used by PAH-degrading
bacteria to enhance their mobility in such a way that
PAH-biodegradation in unsaturated soil is enhanced
(Kohlmeier et al., 2005; Wick et al., 2007).
In the frame of our attempts to develop novel bioremediation approaches based on ecological principles we
tested the hypothesis that fungal networks facilitate the
movement of bacteria by providing continuous liquid films
in which gradients of chemoattractants can form and
chemotactic swimming can take place.
Results
Chemotaxis and motility tests
Chemotactic response was tested in capillaries containing
sodium salicylate. The chemotactic strain PpG7 exhibited
a statistically significant (twofold) attraction to the
chemoattractant relative to the salicylate-free control. The
non-chemotactic derivative G7.C1 showed no chemotactic response to salicylate (P = 0.01). Both strains showed
equal (P < 0.05) swimming and swarming motility on soft
agar plates with colony diameters of 2.3 ⫾ 0.3 and
2.8 ⫾ 0.1 cm obtained after 24 and 48 h on swimming
agar plates and 5.3 ⫾ 0.5 and 6.3 ⫾ 1.1 cm after 24 and
44 h on swarming agar plates respectively.
Effect of salicylate on fungus-mediated dispersal of
Pseudomonas putida
Chemotactic movement of bacteria along fungal networks
was determined in linear arrangements of agar-patches
and gaps (air-filled) separating them (Fig. 1). The ‘test
track’ included a central and two terminal patches of
Inoculation of bacteria
L3
L2
L1
Centre
R1
R2
R3
Sal -/+ :
(-) ………………………………………………… +
Sal +/+ :
+ ………………………………………………... +
Sal -/- :
(-) ……………………....................................... (-)
potato dextrose agar (PDA) and could thus be completely
overgrown by fungal mycelia. These circular patches
framed rectangular patches on which bacterial distributions were determined. High-purity agarose free of
nutrients prevented bacteria from growing there. Chemoeffectors could be placed on the L3 and R3 positions of
the agarose patches and bacterial inocula on the middle
patch. Depending on both the presence and the placement of the chemical (one side, both sides, control without
chemical) this allowed observing and quantifying random
movement and positive or negative chemotaxis of the
bacteria respectively. Three configurations were tested for
each of the strains: 5 mg sodium salicylate was either
placed at position R3 (set-up: Sal -/+), at positions L3 and
R3 (set-up: Sal+/+), or it was omitted (set-up: Sal -/-;
Fig. 1).
In the absence of a fungal mycelium no dispersal of
chemotactic and non-chemotactic bacteria beyond the
gap surrounding, the middle patch was observed (data
not shown). By contrast, about 0.5–1% [ca. 106-107
colony-forming units (cfu)] of the inoculum moved over the
gaps when a mycelium of Pythium ultimum crossed them.
Figure 2 summarizes the distribution of those bacteria
that passed over the gap next to the middle batch within
48 h. In salicylate-free controls, a quasi uniform distribution of strain PpG7 (P < 0.05) in the L3 and R3 positions
was observed (Fig. 2A). Similar results, but with a significant increase in the more distal parts of the agarose
patches, were obtained when salicylate was put on both
sides (Fig. 2B). Salicylate placed in the R3 position clearly
attracted strain PpG7 to the right side (> 70% of the recovered cells, P = 0.0034) with a maximum accumulation in
position R1 (Fig. 2C). Phenanthrene, a substrate of strain
PpG7 not acting as chemoattractant, placed in the same
position did not attract strain PpG7 (not shown). Considerable dispersal, yet no directed movement was observed
for the chemotaxis-deficient strain G7.C1 when salicylate
Fig. 1. Scheme of the experimental set-up to
asses the chemotactic dispersal of P. putida
along mycelia of P. ultimum: a piece of PDA
( ) inoculated with P. ultimum was placed in
the centre of a sterile plastic Petri dish. Two
agarose rectangles ( ) containing 10 mM
PB were placed at an angle of 180° allowing
for a 1 mm air-filled gap between the lateral
agarose and the PDA pieces. At the outer end
of the agarose pieces, PDA agar ( ) was
placed to promote directed fungal growth over
the agarose. The arrows refer to the positions
where 5 mg of solid salicylate was placed on
the mycelia 48 h prior to inoculation with
P. putida on the central PDA in the three
experimental set-ups tested (Sal -/+; Sal+/+;
Sal -/-).
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 12, 1391–1398
Fungal mycelia allow chemotactic dispersal of bacteria 1393
(A)
70
PpG7 (Sal -/-)
60
% of bacteria translocated
% of bacteria translocated
70
50
40
a
a
a
30
a
a
a
20
10
(B)
PpG7 (Sal +/+)
60
50
40
30
b
b
20
L2
L1
R1
R2
R3
L3
L2
Position of the agar
R1
b
R2
R3
c
(C)
Position of the agar
PpG7 (Sal -/+)
70
60
% of bacteria translocated
% of bacteria translocated
L1
b
0
L3
50
40
30
b
b
20
10
a
10
0
70
a
a
a
a
0
G7.C1 (Sal -/+)
(D)
60
50
40
30
a
a
L3
L2
a
a
a
a
L1
R1
R2
R3
20
10
0
L3
L2
L1
R1
R2
R3
Position of the agar
Position of the agar
Fig. 2. Relative distribution of bacteria translocated in the presence of a mycelium of P. ultimum. Heights of the bars sum up to 100% cells
retrieved from locations beyond the gaps around the middle patch (see Fig. 1). Equal letters refer to a statistically equal (P > 0.001) relative
numbers of bacteria (P > 0.001).
A. Chemotactic P. putida PpG7 (NAH7) in the Sal -/- set-up in the absence of salicylate (n = 3).
B. Strain PpG7 in the Sal+/+ set-up in the presence of symmetrically placed salicylate (n = 3).
C. Strain PpG7 in the presence of salicylate (Sal -/- +) (n = 8).
D. Non-chemotactic P. putida G7.C1 (pHG100) in the Sal -/+ (n = 3) set-up in presence of salicylate.
Visualization of fungus–bacteria interactions
Confocal laser scanning microscopy (CLSM) was
employed for visualizing the hypothesized continuous
liquid films forming along the mycelia of P. ultimum
growing through air-filled space. For this purpose P. ultimum was inoculated on a PDA patch placed on a microscope slide in order to let the mycelia overgrow the dry
10.0
Fraction of bacteria translocated (%)
was put on the R3 position (Fig. 2D), on either sides or
without salicylate (not shown). Quantitative balances,
including the cells remaining on the middle patch,
revealed recovery rates of 51 ⫾ 16% and 42 ⫾ 20% of
the inoculated PpG7 in presence (n = 9) and absence
(n = 6) of salicylate (Fig. 3). This indicates that no growthrelated bias on the mobilization had to be expected in
presence of the chemoeffector. There was no significant
influence (P < 0.05) of the chemoeffector on the overall
mobilization rate of both strains tested (Fig. 3).
1.0
0.1
0.0
1
10
100
Fraction of bacteria recovered (%)
Fig. 3. Double logarithmic representation of the fractions of
bacteria recovered relative to the fraction of bacteria passing over
gap around the middle patch in the presence of mycelia of
P. ultimum in the presence of salicylate (filled symbols) and in its
absence (open symbols). Squares and rhomboids represent strains
PpG7 and PpG7.C1 respectively.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 12, 1391–1398
1394 S. Furuno et al.
was further used to visualize the dispersal of P. putida
PpG7 along the filaments P. ultimum. For this purpose the
bacteria were stained with the fluorescent dye (Syto 24),
subsequently, spot inoculated on the fungal filaments and
the bacterial dispersal continuously analysed for 2 h by
simultaneous recording of the reflection signal of the filamentous network and the fluorescent signal of the bacteria. Analysis of the time series recorded demonstrated
that the synaeretic films allowed for bacterial dispersal
along the mycelia of P. ultimum, as depicted in the CLSM
maximum intensity projection in Fig. 4B. Due to experimental hardware/software limitations, however, the
directed dispersal of the bacterial cells along the fungal
filaments could not be quantified.
Translocation of sodium salicylate along the mycelium of
P. ultimum
Chemotaxis requires the development of a chemical gradient. The spatial distribution of sodium salicylate 48 h
after addition to position R3 was analysed. In the presence of P. ultimum, salicylate was found along the entire
network, also beyond both gaps around the middle patch
(Fig. 5). In the absence of a mycelium, sodium salicylate
was detected solely in positions R3 to R1. Degradation
studies showed that P. ultimum was unable to metabolize
sodium salicylate. No decrease of salicylate concentration
was detected and no growth was observable when
P. ultimum was incubated > 7 days with salicylate as
sole carbon and energy source in shaken liquid cultures
(data not shown).
Discussion
Chemotactic navigation along mycelia of P. ultimum
Fig. 4. Confocal laser scanning microscopy showing maximum
intensity projections of P. ultimum filaments growing on clean glass
surfaces. (A) depicts the presence of the synaeretic liquid films
along P. ultimum filaments by an overlay of the reflection and the
FITC-stained fungal filaments. The arrows in the insert indicate the
dimensions of the liquid film (3–4 mm). (B) visualizes the presence
of P. putida PpG7 [stained with the fluorescent dye Syto 24 (in
green)] within the liquid film along the fungal filaments (reflection).
and clean glass surface. Subsequent staining of the
fungal cell surface proteins with fluorescein isothiocyanate (FITC) allowed analysing the gross fungal morphology, whereas an overlay of the reflection and the FITC
signals of the hyphae allowed visualization and assessment of the thickness (3–4 mm) of the synaeretic liquid film
forming along the filaments of P. ultimum (Fig. 4A). CLSM
Fraction of salicylate translocated (%)
Recent studies revealed that fungal hyphae can promote
microbial dispersal and accelerate biotransformation in
60
50
40
30
20
10
0
L3
L2
L1
Centre
R1
Position of the agar
R2
R3
Fig. 5. Average relative distribution (n = 5) of sodium salicylate
quantified 48 h after addition of 5 mg of the chemoeffector at R3 in
the presence (filled bars) and absence (open bars) of P. ultimum.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 12, 1391–1398
Fungal mycelia allow chemotactic dispersal of bacteria 1395
soil (Kohlmeier et al., 2005; Wick et al., 2007). Using
this effect for improving microbial contaminant degradation, however, would benefit from a comprehensive,
mechanistic understanding of the dispersal process.
In this study, we hence tested the hypothesis that
continuous liquid films along fungal mycelia passing
through air-filled space may allow for the build-up of
chemical gradients and promote the chemotactic dispersal of otherwise immobilized bacteria. Sodium
salicylate was selected as known chemoattractant of
PAH-degrading P. putida PpG7 (NAH7) (Velasco-Casal
et al., 2008). Criteria for the choice of salicylate were its
environmental relevance as metabolite and inducer of
microbial PAH-degradation, its high water solubility and
the absence of degradation by P. ultimum. The observed
directed dispersal of strain PpG7 (yet not of its nonchemotactic derivative G7.C1) in the presence of
mycelia of P. ultimum thus suggests that liquid water
films supporting chemotactic swimming formed along the
mycelia. This was unequivocally confirmed by CLSM
visualization clearly showing the liquid film along the
fungal filaments and bacterial motility within such liquid
films respectively. At this point it remains open if the
chemical gradient required for chemotaxis developed
only in extra-hyphal liquid films or if its build-up was also
promoted by the hyphae themselves. The accumulation
of strain PpG7 near the chemoattractant cannot be
explained by advective transport along the hyphae as: (i)
bacteria have to be motile to be transported along P. ultimum (Wick et al., 2007), (ii) the similarly motile but
non-chemotactic derivative G7.C1 showed no preferred
movement to salicylate and (iii) no preferred accumulation of strain PpG7 near growth substrates not acting as
chemoattractant (e.g. phenanthrene) was observed.
Directed navigation to sodium salicylate is in good
agreement with results from classical capillary experiments showing two to fourfold enhanced transport (this
study, Velasco-Casal et al., 2008). No influence of
salicylate on overall bacterial mobility (0.5–1%) was
found (Fig. 3). This may indicate that under non-growth
conditions chemical gradients influence the direction
but not the extent of bacterial swimming. It may also be
that the fraction of transported bacteria represented
basically all those on the middle patch that could get
access to the liquid films in fungal mycelium and make
use of the chemical gradient developing therein. As a
further possibility the capacity of water films to facilitate
active movement of bacteria may simply be limited
and/or further restricted by mechanisms such as
bacterial attachment to hyphae and swimming into
dead ends, etc., although the nearly homogeneous
distribution of bacteria in the Sal +/+ and Sal -/set-up seems to contradict an importance of the latter
mechanisms.
Relevance for the ecology of contaminant
biodegradation
In terrestrial environments, fungi are of fundamental
importance as decomposer organisms and plant symbionts, playing pivotal roles in the carbon, nitrogen and
phosphorous cycles. In most natural soil habitats, the
spatio-temporal distribution of nutrients and minerals is
heterogeneous due to their distinct and complex physical
structures (Boswell et al., 2003). It is thought that many
fungi are able to grow in low-nutrient or polluted habitats
by translocating resources available in other parts of the
mycelium. There is currently evidence of passive translocation (diffusion-driven) and active translocation (metabolically driven) of nutrients (Boswell et al., 2002) by
mycorrhizal fungi. Recent studies have shown that the
underground fungal networks link plants together by
transferring nutrients from plant to plant (Whitfield, 2007)
or provide nutrients to bacteria in their hyphosphere and
hence shape soil microbial communities. As not much is
known about the translocation of substances other than
nutrients in non-mycorrhizal fungi, we compared the
observed translocation of sodium salicylate in the presence of P. ultimum within 48 h with the calculated efficiency of aqueous diffusion. During this time diffusion of
salicylate should be restricted to less than 2 cm. However,
we found significant concentrations of salicylate behind
the gap on the middle patch and minor concentrations of
salicylate on positions L1–3, which is between 1.6 cm and
more than 4 cm away from the source at position R3. As
salicylate is non-volatile, the transport over relatively long
distances may indeed point at a mechanism of intrahyphal
transport.
The observed directed transport of bacteria through
air-filled space suggests that soluble chemicals diffusing
along liquid layers on fungal hyphae can be sensed by
chemotactic bacteria and allow them to rapidly navigate
towards substrate-rich microhabitats. This may be of particular importance for the establishment of plant–microbe
interactions in the rhizosphere or for the spreading of
bacteria in soil pores (Alexandre et al., 2004). Whereas
dispersal of bacteria is generally constrained to matric
potentials above -50 kPa (Paul and Clark, 1996), fungal
subsurface movement is not restricted to fully water-filled
pathways (Ritz, 1995). As fungi are predominantly aerobic
microorganisms that tend to oxidize hydrophobic organics
and break-down polymers by the use of extracellular
enzymes (Ekschmitt et al., 2008), one may speculate that
they do not only render substrates more soluble but also
transfer them to bacterial commensals in the hyphosphere (Johnsen et al., 2005; Wick et al., 2009).
In defined systems, bacterial chemotaxis has been
shown to improve rates of biodegradation (Marx and
Aitken, 2000), given that the rate-limiting factor is the slow
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 12, 1391–1398
1396 S. Furuno et al.
effective mass transfer of the pollutant. Chemotactic dispersal coefficients are two- to three orders of magnitude
greater than random motility coefficients of bacteria (Ford
and Harvey, 2007). From our observations we can conclude that fungal mycelia transform water-unsaturated
porous media into microhabitats, in which bacterial
chemotaxis can function and thus contribute to improved
accessibility of chemicals, such as environmental contaminants. The likely consequence is increased contaminant degradation and easier build-up and maintenance of
contaminant-degrading bacterial communities.
Experimental procedures
Organism and culture conditions
Pseudomonas putida PpG7 (NAH7) (Dunn and Gunsalus,
1973) and its derivative P. putida G7.C1 (pHG100) (Grimm
and Harwood, 1997) were used throughout this study.
Whereas the parent strain PpG7 is known to be chemotactic
towards naphthalene and salicylate, strain P. putida G7.C1
(pHG100) is non-chemotactic (Velasco-Casal et al., 2008).
Further criteria for the choice of these organisms were insignificant differences in motility, as well as in physico-chemical
surface properties and deposition efficiencies in sand-filled
columns (Velasco-Casal et al., 2008). Both strains are motile
by means of polar flagella and able to use naphthalene and
salicylate as the sole carbon and energy source. Naphthalene degradation kinetics of both strains in well-mixed conditions are comparable (Marx and Aitken, 2000). The strains
were grown at 25°C on a gyratory shaker (130 r.p.m.) in
300 ml Erlenmeyer flasks containing 100 ml of liquid minimal
medium (Wick et al., 2001) in the presence of 1.5 g of solid
naphthalene (> 98%, Fluka; crystals taken as obtained by the
provider). Cells used in mobilization experiments were harvested in the late exponential phase after about 48 h of
growth, washed three times in cold 10 mM potassium phosphate buffer (PB) at pH = 7.2, and suspended in 10 mM PB to
obtain a bacterial suspension containing ca. 5 ¥ 109-1 ¥ 1010
cells ml-1. In mobilization experiments, bacteria were quantified as cfu on Luria broth (LB) agar (2% w/v) containing
200 mg l-1 of actidione (cycloheximide). Fungus-associated
bacteria were detached by sequential vortexing (60 s at
40 Hz) and ultrasonication (2 ¥ 30 s) in PB prior to cultivation.
The fast-growing, hydrophilic (Smits et al., 2003; Wick et al.,
2007) oomycete P. ultimum was cultivated at room temperature on solid medium containing 2% (w/v) PDA (Difco) in the
dark.
Swimming and swarming tests
The motility of bacterial strains was tested using a standard
procedure (Rashid and Kornberg, 2000) based on soft agar
plates: swimming agar consisted of (l-1): 10 g of tryptone
(Oxoid), 10 g of yeast extract (Oxoid), 12.5 g of NaCl (Merck)
and 3 g of agar (Difco). Swarming agar was prepared from
(l-1): 8 g of nutrient broth (Oxoid), 5 g of glucose and 5 g of
agar. Freshly poured agar plates were dried for 1 h (swimming agar) and 14 h (swarming agar) at ambient temperature
prior to spot-inoculation with a sterilized toothpick. Motility
was examined by measuring the mean diameter of the bacterial colonies after 24–48 h of incubation.
Degradation of salicylate by P. ultimum
Salicylate biodegradation by P. ultimum was studied by adding
500 ml of a dense mycelium suspension to amber 250 ml
bottles containing 1 g l-1 of sodium salicylate (Merck) dissolved in 100 ml of GASnM medium (Crowe and Olsson,
2001). The fungal suspension was prepared by transferring
PDA-grown mycelia of a 3-day-old P. ultimum to 10 ml GASnM
medium and subsequent vortexing for 60 s at 40 Hz. Samples
without fungal mycelium and/or GASnM medium without
glucose and asparagine, respectively, were used as controls.
Biodegradation of sodium salicylate was quantified for 7 days
by a standard procedure (Trinder, 1954). Briefly, 1 ml of the
analyte solution was mixed with the Tinder’s reagent in the
absence of mercuric chloride and the concentration of sodium
salicylate in iron–salicylate complex was quantified spectrophotometrically at 540 nm. Sodium salicylate standards with
known concentrations were used for calibration.
Capillary chemotaxis experiments
A modified version of the capillary tests described by Adler
(1973) was used to quantify chemotactic response of strain
G7.C1 to sodium salicylate. In short, late exponential cells
were harvested, centrifuged and re-suspended in minimal
medium to an OD578 of ca. 0.020 (corresponding to 106
cells ml-1). About 0.1 ml of this suspension was placed in a
small chamber formed between two capillary tubes placed
parallel to each other on a microscope slide. Another capillary
tube (1 ml volume), heat-sealed at one end, containing the
chemoeffector solution, was immersed in the cell suspension
with its open end. The system was then closed with a glass
coverslip, avoiding any formation of air bubbles in the
chamber. The chemoeffector solution in the capillary contained 10 mM PB supplemented with 100 mM of salicylate.
Minimal medium lacking any chemoeffectors was used as a
control. The chambers were incubated for 1 h at room temperature and the numbers of bacterial cells accumulated in
the test capillaries quantified as cfu on LB agar.
Chemotaxis experiments on fungal networks
Set-up and fungal growth. Chemotactic response of strains
PpG7 and G7.C1 moving along fungal networks towards
chemoeffector gradients was determined in a controlled laboratory system mimicking a combination of water-saturated
(agar surface) and non-saturated (air gap between the agar)
microhabitats (Fig. 1): a patch of PDA (diameter: 1.4 cm,
height: 0.4 cm) inoculated with P. ultimum was placed in the
centre of a sterile plastic Petri dish. Two agarose rectangles
[(l) ¥ (w) ¥ (h): 1.5 ¥ 1 ¥ 0.4 (cm)] containing 10 mM of PB
were placed next to it allowing for a 1 mm air-filled gap
between the agarose and the middle patch. This gap prevented the bacteria from leaving the middle patch in absence
of fungi. For the rectangles, nutrient- and carbon-deficient
agarose (Sigma) was used to limit bacterial growth during the
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 12, 1391–1398
Fungal mycelia allow chemotactic dispersal of bacteria 1397
experiment. At the outer end of each agarose rectangle, a
piece of PDA (diameter: 1.4 cm, height: 0.4 cm) was placed
to promote the growth of fungal mycelium over the entire
arrangement including the agarose rectangles, where no
substrate was present. The Petri dishes were closed and
incubated at the room temperature in the dark. After the
fungus had fully overgrown the system (ca. 5 d), the outer
PDA patches were removed and 5 mg of solid sodium salicylate was added unilaterally at the outer end of one of the
agarose rectangles (position R3 in Fig. 1). Two control experiments were performed by either placing no sodium salicylate
or by placing salicylate at positions L3 and R3 (Fig. 1). Fortyeight hours after the addition of sodium salicylate, 5 ¥ 109
cells m l-1 (as determined as cfu on LB agar) suspended in
5 ml 10 mM PB were applied onto the fungal inoculum on the
middle patch with a sterilized 25 ml microsyringe. Experiments without P. ultimum served as controls to exclude bacterial dispersal to the agarose rectangles in the absence of
fungal hyphae.
Quantification of bacterial transport efficiency. Forty-eight
hours after the addition of the bacteria, their spatial distribution was assessed. The time point was chosen as the time
needed for salicylate to diffuse through the entire rectangle
on which it was placed. The calculation was: with td, L and D
representing the diffusion time (s), the diffusion length (cm)
and the diffusion constant (Dsalicylate = 1.2 ¥ 10-5 cm2 s-1)
(Schwarzenbach et al., 2003). The agarose rectangles were
subsequently cut into three individual sections of 0.5 cm
length. Each of them, as well as the middle patch, were
placed in sterile glass test tubes containing 2 ml 10 mM PB.
Surface-associated bacteria were subsequently detached
from the fungal mycelia and the agar as described above and
quantified as cfu on LB agar containing 200 mg l-1 of actidione (cycloheximid) to suppress fungal growth. Distribution of
salicylate on the agarose after 48 h was measured using the
Trinder method as described above.
Confocal laser scanning microscopy
The CLSM observations were performed with a TCS SP1
(Leica) attached to an upright microscope equipped with a
63¥ NA 0.7 air and 63¥ NA 0.9 water immersion objectives.
The instrument was controlled by the Leica Confocal Software Version 2.61 Build 1537. For excitation the laser lines at
488, 561 and 633 nm were available. The microscope was
used for imaging the transmission, reflection, autofluorescence and fluorescence signals. The samples were screened
for protein, polysaccharides, nucleic acids, lipids and lectin
binding (Staudt et al.) stains. In Fig. 4A the fungal filament
surfaces were stained with FITC (Research Organics) and in
Fig. 4B the bacterial nucleic acids with Syto 24 (Invitrogen).
The sample was excitated at 488 nm, and emission signals
were detected at 480–500 nm (reflection) and 500–550 nm
(FITC and Syto 24). Image data were projected using the
microscope software and Imaris 6.3 (Bitplane). In order to
improve signal to noise ratio, the data set in Fig. 4A was
subjected to blind deconvolution using the classic MLE algorithm in Huygens 3.0.0 (SVI). Images were finally printed from
Photoshop (Adobe) without any further adjustments.
Statistical analysis
Unless otherwise stated, all P-values are < 0.05 as determined based either on Student’s t-test or one-way ANOVA.
Acknowledgements
This study was supported by the European MC-EST 20984
grant (RAISEBIO). It contributes to the CITE research programme of the Helmholtz Association. Technical help by R.
Remer, B. Würz, J. Reichenbach, M. Kolbe, K. Lübke and U.
Kuhlicke is greatly acknowledged. We further thank Prof. C.
S. Harwood (University of Washington) for the provision of
P. putida G7.C1 (pHG100).
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