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Applied and Environmental Microbiology, May 2001, p. 2088-2094, Vol. 67, No. 5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.5.2088-2094.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Induction of Laccase Activity in Rhizoctonia solani by
Antagonistic Pseudomonas fluorescens Strains and a Range of
Chemical Treatments
Jonathan D.
Crowe and
Stefan
Olsson*
Section of Genetics and Microbiology,
Department of Ecology, The Royal Veterinary and Agricultural
University, DK-1871 Frederiksberg, Copenhagen, Denmark
Received 11 December 2000/Accepted 26 February 2001
 |
ABSTRACT |
Fungi often produce the phenoloxidase enzyme laccase during
interactions with other organisms, an observation relevant to the
development of biocontrols. By incorporating the laccase substrate 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) into agar,
we analyzed laccase induction in the plant-pathogenic fungus Rhizoctonia solani when paired against isolates of the soil
bacterium Pseudomonas fluorescens. Substantial induction of
R. solani laccase was seen only in pairings with strains of
P. fluorescens known to produce antifungal metabolites. To
study laccase induction further, a range of chemical treatments was
applied to R. solani liquid cultures.
p-Anisidine, copper(II), manganese(II), calcium ionophore
A23187, lithium chloride, calcium chloride, cyclic AMP (cAMP),
caffeine, amphotericin B, paraquat, ethanol, and isopropanol were all
found to induce laccase; however, the P. fluorescens metabolite viscosinamide did not do so at the concentrations tested. The stress caused by these treatments was assessed by measuring changes
in lipid peroxidation levels and dry weight. The results indicated that
the laccase induction seen in pairing plate experiments was most likely
due to calcium or heat shock signaling in response to the effects of
bacterial metabolites, but that heavy metal and cAMP-driven laccase
induction was involved in sclerotization.
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INTRODUCTION |
Rhizoctonia solani is a
soil-living plant-pathogenic fungus which attacks a wide range of crop
plants, including sugarbeet, potato, and rice. Seeds and seedlings are
particularly susceptible to this fungus, which persists in soil in the
form of resistant sclerotia. Fungicides can be used to control R. solani, but recently attention has focused on the development of
biocontrol techniques. To this end, strains of the bacterium
Pseudomonas fluorescens that are antagonistic to R. solani have been isolated and are currently being evaluated as
biocontrol agents in our department. The most promising strain is
P. fluorescens DR54, which produces the antifungal
depsipeptide metabolite viscosinamide (18).
Inevitably, most fungi will encounter competitive or antagonistic
organisms, whether bacterial, fungal or animal, during their life
cycles. Fungi engaged in such competition frequently produce secondary
metabolites, extracellular phenol-oxidizing enzymes, and differentiated
structures in the zone of conflict (2, 4, 8). These
responses may be critical in determining the outcome of a biocontrol treatment.
Our first aim was to study the induction pattern of the fungal
phenol-oxidizing enzyme laccase in interactions between R. solani and P. fluorescens and how this related to the
characteristics of different P. fluorescens strains.
Laccase, ubiquitous in fungi and flexible in function
(28), is often induced during antagonistic interactions,
and R. solani has been shown to possess four laccase genes
(29). Our second aim was to determine the pathways of laccase induction in R. solani and relate these to the
effects of antagonistic bacteria. We believed that this work would aid the development of the biocontrol system and provide insights into the
physiology and ecology of the two organisms.
| |
MATERIALS AND METHODS |
Media and chemicals.
Potato dextrose agar (PDA), glucose,
and asparagine were obtained from Difco Laboratories. All other
chemicals used were obtained from Sigma, apart from high-pressure
liquid chromatography purified viscosinamide, which was provided
in-house by Tommy H. Nielsen.
Organisms.
R. solani AG4 (strain 92009; Danisco
Seed, Holeby, Denmark) was maintained on PDA. The growth conditions
used were 25°C in darkness for both agar and liquid cultures.
P. fluorescens strains were previously isolated from an
experimental field of Danisco Seed and maintained in our departmental
culture collection (18). The strains used were 96.578, DR1, DR2, DR3, DR4, DR12, DR17, DR20, DR34, DR41, DR46, DR48, DR50,
DR52, DR54, DR56, PS1, PS3, PS7, PS8, PS12, PS16, and PS21. Several of
these strains produce antifungal metabolites. DR54 produces the
depsipeptide viscosinamide, similar in structure to the surfactant
compound viscosin (14) but less polar. PS8 and PS16
produce 2,4-diacetylphloroglucinol (DAPG); PS7 and 96.578 produce,
respectively, cyclic and noncyclic isoforms of a peptide similar to
viscosinamide and tentatively named tensin (T. H. Nielsen,
unpublished data). DR50 produces an uncharacterized antifungal
metabolite, extractable by ethyl acetate (Nielsen, unpublished).
Pairings of R. solani and P. fluorescens.
Pairings were made between all P. fluorescens strains and R. solani. Agar plugs 4 mm in
diameter were taken from an R. solani culture and
transferred, approximately 2 cm off center, to PDA plates. Two parallel
inoculation streaks of the P. fluorescens strain were then
made facing the R. solani inoculation plug (see Fig 2A). To
ensure reproducibility, inocula were positioned on the plates by
reference to a drawn template. Three replicates were made of each
pairing, and the experiment was repeated twice.
ABTS underlay visualization of laccase production.
The
artificial laccase substrate
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was used
to visualize laccase distribution in agar cultures of R. solani. Indicator agar was made as follows. A mixture of 0.5487 g
of 1 mM ABTS, 20 g of agar, and 50 ml of 20× mineral medium (per
liter, 2 g each of K2HPO4, KCl, and
MgSO4 · 7H2O 2.0 [pH 5.0]) was made to
1 liter with distilled water and autoclaved; 20-ml aliquots of
indicator agar were pump dispensed into 9-cm-diameter plastic petri
dishes. Laccase distribution in an agar culture was visualized by
removing the colonized agar from its dish and laying it on top of a
plate of indicator agar. Bubbles were excluded by gentle pressure from
a gloved finger, and the plate was incubated for 120 min at room
temperature. ABTS oxidized by laccase became visible as dark green
regions on the indicator agar. The colonized agar was discarded, and
the underlay agar then photographed on a light box using a 35-mm camera
and Kodak Gold 200 print film. The contribution of peroxidase activity to color development was assessed by cutting agar from regions close to
the interaction zone, extracting in Britton-Robinson buffer (0.1 M
boric acid-0.1 M acetic acid-0.1 M phosphoric acid adjusted to pH 5.0 with 0.5 M NaOH), and measuring ABTS oxidation in the presence of
catalase (1 mg/ml) (see description of laccase assay method below).
Image analysis procedure.
The photographic prints were
digitized to produce 256-level greyscale images of approximately 1,800 pixels square. The Magic Wand function of Paint Shop Pro software
(shareware version 4.12; JASC Inc.) was used to select regions within
each plate that were at least 10 greyscale points darker than the
background agar, which was deleted. The area, in pixels, of the dark
regions was measured by using the Area function of Image-Pro Plus
software (version 1.2; Media Cybernetics L.P.). The image of the petri dish was outlined using a circular selection tool, and its pixel area
was measured. The area of visible ABTS oxidation was then expressed as a fraction of total petri dish area.
Liquid culture of R. solani.
R.
solani was grown in a defined liquid medium modified from GAsnM
(19). This contained (in grams per liter) glucose (9.0), asparagine (1.0), K2HPO4 (0.1), KCl (0.1),
MgSO4 · 7H2O (0.1), and thiamine
(10
3); 25 µl of a trace element solution was also
added, consisting of (in grams per liter) FeEDTA (34.4),
ZnSO4 · 7H20 (6.3),
MnSO4 · H2O (15.4),
CuSO4 · 5H2O (2.5), and
NH4Mo7O2 · 4H2O
(0.5). R. solani inoculum was prepared by plug inoculating a
PDA plate covered with a cellophane membrane and incubating it at
25°C for 3 days. The mycelium was scraped from the plate using a
sterile glass microscope slide, added to 10 ml of 0.3 M mannitol, and
macerated for 1 min in a Sorval Omni-Mixer fitted with a 50-ml beaker.
Conical flasks (300 ml) containing 40 ml of GAsnM medium were each
inoculated with 400 µl of mycelial suspension. The flasks were
incubated in the dark at 25°C without shaking.
Chemical treatment of R. solani cultures.
Chemical treatments were added to the liquid cultures of R. solani after 48 h of growth. Each experiment used the same
batch of R. solani inoculum in two to four sets of five
replicate flasks. A set of five control flasks was included in each
experiment. The chemicals were chosen to treat the R. solani
liquid cultures were either (i) known or suspected to induce fungal
laccases as substrates or cofactors, (ii) likely to affect
intracellular message pathways controlling gene regulation, or (iii)
known to cause stress to the fungus by various means. The final
concentration of each chemical used (see Fig. 3) was determined either
by reference to available literature or preliminary experiments
(indicated as "P"). The chemicals chosen were amphotericin B (P),
p-anisidine (P), caffeine (23),
CaCl2 (P), calcium ionophore A23187 (P), CuSO4
(P), cyclic AMP (cAMP) (23), ethanol (P), isopropanol (P),
LiCl (13), MnSO4 (P), paraquat (methyl
viologen) (P), and viscosinamide (C. Thrane, unpublished data). In
cases where ethanol was used as a solvent (p-anisidine,
calcium ionophore A23187, and viscosinamide), the same volume of
ethanol was applied to control cultures. As the effects of
CaCl2 and viscosinamide were small, these experiments were
repeated three times.
Assay of laccase activity in liquid cultures.
Five laccase
assays of the culture medium were carried out at approximately 24-h
intervals. The first assay was made immediately prior to treatment of
the cultures. The assay buffer used was a mixture of 0.1 M boric acid,
0.1 M acetic acid, and 0.1 M phosphoric acid (Britton-Robinson buffer
[29]) adjusted to pH 5.0 with NaOH. A 200-µl sample of
culture medium was mixed with 750 µl of assay buffer in a 1.6-ml
semimicrocuvette. To this was added 50 µl of a 20 mM aqueous solution
of ABTS (extinction coefficient of 35 mM1
cm1 at 405 nm). The increase in absorbance at 405 nm was
recorded over 1 min on a Shimadzu UV-160 UV-visible recording spectrophotometer.
LPO assay and dry weight measurement.
The Bioxytech LPO-586
lipid peroxidation (LPO) assay (Oxis International) was used to measure
LPO products including 4-hydroxyalkenals and malonaldehyde. LPO and dry
weight measurements were carried out approximately 200 h after
inoculation. Mycelium from liquid cultures was harvested, blotted dry
on paper towels, and then ground for 30 s in a mortar and pestle
with 10 ml of distilled water; 1.5 ml of this sample was centrifuged at
10,000 × g for 15 min to remove debris; 200 µl of
the supernatant was assayed for both malonaldehyde and
4-hydroxyalkenals according to the manufacturer's protocol. The dry
weight of the mycelium was calculated by pouring the mycelial slurry
into preweighed petri dishes and drying overnight at 55°C before reweighing.
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RESULTS |
Laccase induction in confrontations between R. solani
and a panel of P. fluorescens strains.
Twenty-three
strains of P. fluorescens were tested for the ability to
induce laccase activity in R. solani. These strains had
previously been assessed for inhibitory effects on R. solani and screened for the production of pigments, endochitinases, and antifungal metabolites (18). None of the P. fluorescens strains were able to oxidize ABTS directly. The
fractional area of the interaction plates where ABTS oxidation could be
detected using image analysis is presented in Fig.
1. Of the 23 P. fluorescens strains tested, 17 induced the formation of measurable regions of
laccase activity in interactions with R. solani. Of the
remaining strains, three induced trace amounts of laccase and three
caused no induction. The only P. fluorescens strains to
induce laccase in more than 2% of the plate area were those known to
produce antifungal agents. No positive correlation was seen between
R. solani laccase induction and P. fluorescens
strains expressing endochitinase or producing pigment. Assays of agar
extracts did not show reduced ABTS oxidation in the presence of
catalase, indicating that peroxidases were not involved.
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FIG. 1.
Laccase response of R. solani to various
P. fluorescens strains in agar cultures. The boxed letters
refer to metabolites known to be produced by certain strains of
P. fluorescens. D, DAPG; T1, tensin (cyclic); T2, tensin
(linear); X, uncharacterized (ethyl acetate extractable); V,
viscosinamide. The strains of P. fluorescens not inducing
measurable areas of laccase activity were DR12, DR20, DR46, DR56, PS12,
and PS21.
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The pattern of ABTS oxidation seen in the
R. solani-
P.
fluorescens DR54 interaction illustrated in Fig.
2 is typical of all
cases where
substantial laccase activity was observed, being localized
to regions
at the edge of the fungal colony where growth inhibition
was evident.
In some cases, small, sharply defined areas of low
laccase activity
appeared within the high-activity regions. Sclerotial
formation was
limited and did not correlate with regions of laccase
activity. In
interactions involving DAPG-producing strains of
P. fluorescens, the region of ABTS oxidation (and no other regions)
initially appeared orange, although further development of green
coloration gradually masked this.
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FIG. 2.
Induction of R. solani laccase by P. fluorescens DR54 on 9-cm-diameter PDA plates (3 days). R. solani shows appressed and inhibited mycelial growth near streaks
of P. fluorescens DR54 (A). Laccase activity was visualized
using an ABTS agar underlay. R. solani laccase activity was
induced in the region of confrontation (B).
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|
Induction of laccase in liquid cultures of R. solani by
chemical treatments.
The liquid cultures of R. solani
initially grew as a pale diffuse submerged mycelium, producing the
first aerial growth after 36 to 48 hs and developing into a floating
mycelial mat. Sclerotization was limited (<5% of mycelial surface)
and commonly occurred in contact with the flask walls. In untreated
cultures, laccase activity peaked at about 96 h, concomitant with
the onset of yellow-brown pigmentation and the early stages of
sclerotial formation. The mean peak laccase activity of control
cultures (data pooled from all experiments) was 8.6 U
ml1, with a standard error of the mean of ±2.
The induction profiles of the liquid culture laccase assays are
presented in Fig.
3, and the relative
peak inductions are
given in Table
1.
Treatments with all of the chemicals except
viscosinamide induced
laccase to some extent. In most cases, the
peak induction of laccase
occurred at or after 48 h posttreatment.
The exceptions were
ethanol and isopropanol, with peak activity
appearing by 24 h.
cAMP inhibited laccase production at the first
time point posttreatment
(
P < 0.001), with induction delayed until
72 h
posttreatment. The most potent inducer of laccase was the
calcium
ionophore
A23187, which induced laccase more than 13-fold
at a
concentration of 5.7 µM.
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FIG. 3.
Profiles of laccase activity in liquid cultures of
R. solani treated with various chemicals. One unit of
activity is the amount of enzyme converting 1 µmol of substrate
in 1 min. Each point is the mean of five replicates with SEM. Cont.
(Et.), control (ethanol); Ampho. B, amphotericin B.
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TABLE 1.
Effects of chemical treatments on laccase induction,
final dry weight, and LPO levels in liquid cultures of R. solania
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|
Stress measurements and morphological changes in chemically treated
cultures.
The LPO levels, dry weights, and morphological changes
seen in chemically treated cultures are presented in Table 1.
Correlated stress effects (LPO increase and dry weight decrease) were
seen in caffeine, CuSO4, isopropanol, and paraquat
treatments. Ethanol and amphotericin B increased LPO but did not reduce
dry weight. Measurements of dry weight in older cultures indicated that
R. solani could metabolize ethanol (data not shown). LiCl
treatment reduced dry weight, but its effect on LPO could not be
assessed because it interfered with the assay. cAMP reduced LPO and dry weight, and CaCl2 reduced LPO but left dry weight
unaffected; both of these treatments may have antioxidant effects that
limit LPO (see Discussion). Calcium ionophore A23187,
p-anisidine, MnSO4, and viscosinamide did not
cause significant effects.
Effects of the chemical treatments on the morphology of liquid cultures
varied. Ethanol, isopropanol, and paraquat abolished
pigmentation and
sclerotial formation, whereas copper, manganese,
caffeine, and cAMP
caused heavy sclerotization with varying degrees
of pigmentation.
Amphotericin B-treated cultures developed a pale,
felty mycelial mat
containing many loose, pigmented spots resembling
flattened sclerotia.
Neither
p-anisidine or LiCl altered the sclerotization
of
cultures, but both caused deep pigmentation of the mycelium
and culture
medium. Viscosinamide treatment inhibited aerial mycelium
formation at
the periphery of the mycelial mat, possibly due to
its surfactant
properties.
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DISCUSSION |
Laccase induction in pairing plates.
We found that several
strains of P. fluorescens known to produce antifungal
metabolites were able to induce substantial laccase production by
R. solani. This was not linked to mycelial melanization or
sclerotization, processes often associated with laccase. The pattern of
induction suggested that the R. solani mycelium was reacting
to agents diffusing from the bacteria within the agar medium. Both
induction and inhibition of phenoloxidase activity have been reported
in other fungai-bacterial interactions. Bacillus subtilis
cells and extracts were found to induce laccase in the wood-decaying
basidiomycete Hypholoma fasciculare (8), and Pseudomonas tolaasii enhanced tyrosinase activity in
Agaricus bisporus fruit bodies by the action of a metabolite
resembling viscosinamide (27). Exposure of the
mycoparasite Phanaerochaete magnoliae and saprophyte
Trichoderma viride to growth-inhibiting volatiles from soil
bacteria inhibited laccase and induced or inhibited tyrosinase,
depending on the bacterial strain (16). Our results show a
great variability in laccase response to different strains of the same
bacterial species, with the key inducing factor in this case being
antifungal metabolite production. This implies scope for great
complexity in the biochemical interplay between fungi and bacteria in
the soil community.
Potential control pathways for R. solani laccases.
The results of the chemical treatment experiments shown in Table 1 and
Fig. 3 indicate that several signaling pathways may be involved in
laccase regulation in R. solani, and a hypothetical scheme
for this is presented in Fig. 4.
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FIG. 4.
Hypothetical scheme for laccase induction in R. solani. Thin arrows refer to the direct or indirect effects of
different chemical treatments; heavy arrows indicate endogenous
signaling pathways. Treatments marked * may have a minor oxidative
stress effect. A23187 refers to the calcium ionophore; Am-B denote
amphotericin B.
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Calcium is a ubiquitous secondary messenger in fungi (
6)
and has been directly linked to laccase induction in the fungus
Cryphonectria parasitica (
13). Treatment of
R. solani liquid
cultures with calcium ionophore
A23187,
amphotericin B, or
lithium chloride all induced laccase activity. All
of these compounds
mobilize calcium across membranes by various means
(
21,
25).
The effect of the ionophore was particularly
significant, as it
was highly potent and not associated with stress
effects or alterations
in mycelial morphology. The lithium ion acts
indirectly on calcium
levels by stimulating the release of calcium from
intracellular
stores via inositol 1,4,5-trisphosphate-sensitive calcium
channels
(
6,
13); the lithium ion-triggered induction of
laccase seen
here suggests that such a pathway functions in
R. solani. Finally,
although intracellular calcium levels are thought
to be tightly
controlled (
6), addition of calcium chloride
to the growth
medium also led to a modest laccase
induction.
cAMP is another important secondary messenger known to drive many
processes including sclerotial formation in
R. solani
(
26)
and other fungi (
6,
23). We found
R. solani laccase activity
and sclerotization to be strongly
induced by both cAMP itself
and caffeine, an antagonist of cAMP
phosphodiesterase. These results
point to the regulation of laccase by
cAMP and the involvement
of both in sclerotial morphogenesis
(
28). Laccase activity concomitant
with sclerotization was
also seen after both Cu
2+ and Mn
2+ treatments.
Copper is both a cofactor (
28) and a transcriptional
inducer of fungal laccases (
5), and the redox cycling of
manganous
ions is indirectly driven by laccase during ligninolysis
(
17).
Our results imply that a metal-responsive induction
pathway is
involved in laccase induction and further strengthen the
link
between laccase and
sclerotization.
The finding that ethanol and isopropanol are powerful laccase inducers
is, to our knowledge, novel. This induction was rapid
(<24 h) compared
to all other treatments, and isopropanol induced
the highest recorded
laccase activity. Alcohols destabilize membrane
and protein structures
(
10), and the cellular response to this
is closely
correlated with that of heat shock (
20), which regulates
laccase and peroxidase genes in some fungi (
15,
24).
Calcium
influx and oxidative stress (due to membrane disruption and
metabolic
by-products, respectively) are also likely consequences of
alcohol
treatment. As isopropanol is more lipophilic than ethanol, it
is correspondingly more disruptive, as reflected in its greater
laccase-inducing potential. This intense, rapid inducing activity
could
provide a useful tool for further investigations of laccases
in
R. solani and other
fungi.
The aromatic compound
p-anisidine is known to induce laccase
in
Rhizoctonia species (
29), and such specific
induction may
operate via receptor-mediated transcriptional activation
(
5).
As expected, we found
p-anisidine to be a
powerful inducer of
laccase in
R. solani, and this induction
was not linked to any
stress effects at the concentration
used.
It has been proposed that laccase induction (
5,
23; M. Jaszek and A. Leonowicz, Abstr. Int. Conf. Plasma Membr. Redox
Syst. Role Biol. Stress Dis., p. 49, 1998) and sclerotization
(
7) respond to oxidative stress. This may be directly
sensed
via a transcriptional activator or cause indirect activation of
calcium and heat shock pathways due to membrane disruption and
protein
misfolding. Copper, paraquat, and alcohol treatments are
known to cause
oxidative stress via well-established mechanisms
of free radical
formation (
9,
10), and we found that these
treatments
caused increased LPO and dry weight loss as well as
laccase induction.
Although copper and the alcohols have other,
parallel effects (see
above), a straightforward link between oxidative
stress and laccase
induction can be seen in the case of paraquat
treatment. This link
complicates the interpretation of some of
the other chemical treatments
(particularly caffeine and amphotericin
B), as oxidative stress may be
part of their laccase-inducing
effect, a possibility overlooked in most
previous studies. However,
our evidence for the calcium, metal, and
cAMP pathways includes,
in each case, at least one treatment that does
not cause oxidative
stress as measured by LPO. In general, no clear
correlation was
evident between elevated LPO levels and enhanced
sclerotization.
The alcohol treatments increased LPO but actually
inhibited sclerotization
and pigmentation. Alcohols have a paradoxical
effect; although
their metabolic breakdown is a powerful source of free
radicals,
they also scavenge free radicals in solution, and this may
interfere
with melanogenesis (
9).
Unexpectedly, calcium chloride and cAMP treatments were found to
significantly decrease LPO. Calcium has been reported to
protect yeast
cells from Fe
2+ and Cu
2+ toxicity, probably by
competing with these ions at the cell surface
and thus limiting the
production of hydroxyl radicals (
9,
12).
As well as
reducing LPO, cAMP also caused an initial inhibition
in laccase
activity and may act as an antioxidant in addition
to its direct
intracellular signaling
effect.
Viscosinamide was the only
P. fluorescens metabolite
purified in sufficient amounts for experimental use. The metabolite did
not significantly induce laccase in liquid cultures, although
it should
be noted that the hydrophobicity of viscosinamide limited
the
concentration that could be applied without the excessive
use of
solvents. Viscosinamide's potency may be amplified on the
pairing
plates due to highly localized concentration effects and
the
environment (e.g., nutrient depletion) caused by the presence
of live
bacteria.
Significance of laccase induction to the biocontrol and virulence
of R. solani.
We conclude from the evidence discussed
above that the strong laccase induction seen in some R. solani- P. fluorescens interactions is most
likely due to the triggering of calcium and/or heat shock signaling
pathways by bacterial metabolites. Involvement of the substrate-,
metal-, cAMP-responsive pathway seems improbable, as the known P. fluorescens metabolites are unlikely laccase substrates, Cu2+ and Mn2+ concentrations were low, and
sclerotia (associated with cAMP elevation) were absent.
What is the role of laccase in the fungal-bacterial interaction?
Calcium influx and heat shock pathway activation are both
indicators
that cell integrity is compromised, a potentially fatal
stress.
Reactions involving laccase-derived free radical products
may restore
homeostasis in two ways: by polymerizing and rendering
cell walls less
permeable (
24) and/or by detoxifying antifungal
compounds
(
1). Laccase may therefore be a determining factor
in the
efficacy of the bacterial biocontrol of
R. solani. Moreover,
it could play a similar role as a virulence factor in the host-fungus
interaction, a role in which laccase has already been implicated
(
11,
13,
30). Further work is being undertaken to study
which
R. solani laccases are involved, their regulation, and
how
they act in soil or host
systems.
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ACKNOWLEDGMENTS |
We are grateful to Tommy H. Nielsen for valuable information and
help in investigating P. fluorescens metabolite production and to Mette Nielsen and Dorte Rasmussen for supplying and preparing P. fluorescens strains.
This research was supported by grant 9601035 (Functional Specialisation
of the Mycelium) from the Danish Agricultural and Veterinary Research Council.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: The Royal
Veterinary and Agricultural University, Department of Ecology, Section
of Genetics and Microbiology, 40, Thorvaldsensvej, DK-1871
Frederiksberg, Copenhagen, Denmark. Phone: (45) 35282646. Fax: (45)
35282606. E-mail: stefan.olsson{at}ecol.kvl.dk.
| |
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Applied and Environmental Microbiology, May 2001, p. 2088-2094, Vol. 67, No. 5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.5.2088-2094.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
