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Applied and Environmental Microbiology, October 2000, p. 4305-4314, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cytological Effects of Cellulases in the Parasitism
of Phytophthora parasitica by Pythium
oligandrum
Karine
Picard,1
Yves
Tirilly,1 and
Nicole
Benhamou2,*
Laboratoire de Microbiologie et
Sécurité Alimentaire, Université de Brest,
Technopôle Brest-Iroise, 29200 Plouzané,
France,1 and Recherche en Sciences de la
vie et de la santé, Pavillon Charles-Eugène Marchand,
Université Laval, Sainte-Foy, Québec, Canada, G1K
7P42
Received 19 January 2000/Accepted 27 July 2000
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ABSTRACT |
The ubiquitous oomycete Pythium oligandrum is a
potential biocontrol agent for use against a wide range of pathogenic
fungi and an inducer of plant disease resistance. The ability of
P. oligandrum to compete with root pathogens for
saprophytic colonization of substrates may be critical for pathogen
increase in soil, but other mechanisms, including antibiosis and enzyme
production, also may play a role in the antagonistic process. We used
transmission electron microscopy and gold cytochemistry to analyze the
intercellular interaction between P. oligandrum and
Phytophthora parasitica. Growth of P. oligandrum towards Phytophthora cells correlated with
changes in the host, including retraction of the plasma membrane and
cytoplasmic disorganization. These changes were associated with the
deposition onto the inner host cell surface of a cellulose-enriched material. P. oligandrum hyphae could penetrate the
thickened host cell wall and the cellulose-enriched material,
suggesting that large amounts of cellulolytic enzymes were produced.
Labeling of cellulose with gold-complexed exoglucanase showed that the integrity of the cellulose was greatly affected both along the channel
of fungal penetration and also at a distance from it. We measured
cellulolytic activity of P. oligandrum in substrate-free liquid medium. The enzymes present were almost as effective as those
from Trichoderma viride in degrading both carboxymethyl cellulose and Phytophthora wall-bound cellulose. P. oligandrum and its cellulolytic enzymes may be useful for
biological control of oomycete pathogens, including
Phytophthora and Pythium spp., which are
frequently encountered in field and greenhouse production.
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INTRODUCTION |
The potential of fungal antagonists
for biological control of plant pathogens has been recognized for many
years (11), and our understanding of the coordinated series
of events leading to successful parasitism has increased recently
(12, 13). Antagonistic fungi vary greatly in their modes of
action, virulence, and degrees of host specificity (6, 7, 9)
and may use mechanisms such as competition (31), antibiosis
(20), production of hydrolytic enzymes (21), or a
combination of all these processes (12, 28, 30) to attack
their target hosts.
The ubiquitous oomycete Pythium oligandrum Dreschler is a
potential biocontrol agent for use against a wide range of economically important soilborne plant pathogens (1, 10, 15, 26). Detailed information regarding the mechanisms that this fungus uses to
induce plant protection is not available, although, in addition to its
antimicrobial properties, it may be able to induce resistance in host
plants (8). The antagonism exerted by P. oligandrum is thought to involve the action of cell wall
hydrolytic enzymes and/or antibiotics and to depend upon the target
host species (9).
The role played by hydrolytic enzymes in the antifungal activity
attributed to P. oligandrum needs clarification, although a
role for these lytic enzymes in cell wall penetration and degradation often has been suggested (26). The correlation between lytic enzyme synthesis and biocontrol activity by P. oligandrum
remains controversial. For example, isolates of several parasitic
Pythium spp. (17) cannot produce cellulases in
culture media containing cellulose or carboxymethyl cellulose (MC)
(28). Recently, Benhamou et al. (9) showed that
cellulose hydrolysis was a key component of the mycoparasitism of two
pathogenic Pythium spp. by P. oligandrum and that
the isolate of P. oligandrum which they used could
synthesize cellulases which facilitated the mycoparasitic process.
These observations raise several important questions. For example, are
cellulases involved in antagonism of other oomycetes (e.g.,
Phytophthora parasitica)? What is the relative importance of
cellulases and antibiotics? How does the activity of the P. oligandrum-produced cellulases compare with that of similar
enzymes from other sources?
We studied the in vitro interaction between P. oligandrum
1010 and P. parasitica ultrastructurally and cytochemically.
The objectives of the present study were (i) to determine the sequence of events involved in the P. oligandrum-P. parasitica
interaction, (ii) to confirm the involvement of cellulases in the
process resulting in internal colonization of the host, and (iii) to
determine if the activity of these enzymes in terms of
Phytophthora cell wall degradation, was similar to the
activity of the living antagonist. We found that in vitro attack by
P. oligandrum induces a set of events that adversely affects
Phytophthora growth and development. Knowledge of these
events can be used to identify cropping systems and disease management
strategies in which P. oligandrum is likely to function as
an effective biological control agent.
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MATERIALS AND METHODS |
Fungal culture and growth conditions.
P. oligandrum
Drechsler isolate 1010 was recovered from pea roots in Denmark, and
P. parasitica 149 was selected for its virulence on tomato.
Both fungi were grown either on potato dextrose agar (PDA) (Difco
Laboratories, Detroit, Mich.) or on V8 agar (200 ml of V8 juice
[Campbell Soup Company Ltd., Toronto, Ontario, Canada), 2.5 g of
CaCO3, 0.1 g of 
-sitosterol, 15 g of agar, 800 ml of deionized water). Plates were incubated at 24°C in the dark and
were subcultured every week. V8 agar was used for dual-culture test experiments.
For cellulase activity analysis, P. oligandrum was grown in
Erlenmeyer flasks containing 100 ml of Difco potato dextrose broth (PDB) at 24°C for 6 days at 150 rpm. Mycelium was harvested by centrifugation at 7,000 × g for 20 min. The
supernatant was filtered through a 0.2-µm-pore-size filter (Millipore
Corporation, Bedford, Mass.) and stored at 
20°C until it was used
for enzyme analysis.
Dual-culture tests.
The P. parasitica-P.
oligandrum interaction was studied as previously described
(6). Briefly, a PDA disk (diameter, 5 mm) from the margin of
an actively growing P. parasitica culture was transferred to
fresh agar medium. Forty-eight hours later, a plug (diameter, 5 mm) of
P. oligandrum mycelium was placed 3 cm from the P. parasitica disk, and the plates were incubated at 25°C under
continuous light. P. parasitica mycelium began to be
overgrown by hyphae of P. oligandrum 3 days after
inoculation. Samples from the interaction regions taken 1, 2, 3, 4, and
5 days after inoculation of P. oligandrum were processed for
electron microscopy.
Tissue processing for ultrastructural observations.
Mycelial
samples (2 mm3), collected from the region of interaction
between P. oligandrum and P. parasitica, were
fixed by immersion in glutaraldehyde and osmium tetroxide, dehydrated
in a graded ethanol series (25 to 100% ethanol), and embedded in Epon
812 (JBEM Chemical Co., Pointe-Claire, Québec, Canada) as
previously described (6). Ultrathin sections (thickness, 0.1 µm), collected on nickel grids by using a diamond knife, were either
contrasted with uranyl acetate and lead citrate for immediate
examination with a 1200 EX transmission electron microscope (JEOL,
Tokyo, Japan) operating at 80 kV or processed further for cytochemical labeling. An average of five samples from five different plates for
each sampling time were examined. For each sample, 10 to 15 ultrathin
sections were observed.
Cytochemical labeling.
Colloidal gold with particles
averaging 12 nm in diameter was prepared as described by Frens
(18) by using sodium citrate as a reducing agent. The
beta-1,4-exoglucanase-gold complex used for localization of the cell
wall-bound cellulose was prepared as described by Benhamou et al.
(5) by using a beta-1,4-D-glucan cellobiohydrolase (EC 3.2.1.21) complexed to colloidal gold at pH 9.0.
Ultrathin sections were floated for 5 min on a drop of 10 mM sodium
phosphate-buffered saline (pH 6.0) containing 0.02% (wt/vol)
polyethylene glycol 20,000 (Fisher Scientific, Nepean, Ontario,
Canada), transferred to a drop of the exoglucanase-gold-complex,
and
incubated for 30 min at room temperature in a moist chamber.
Grids
carrying sections were washed thoroughly with phosphate-buffered
saline
(pH 7.4), rinsed with distilled water, and allowed to dry
before
staining with uranyl acetate and lead
citrate.
Labeling specificity was assessed with the following controls: (i)
addition of beta-1,4-glucan from barley (1 mg/ml) prior
to section
labeling for a competition experiment; (ii) replacement
of the
enzyme-gold complex being studied by a bovine serum albumin-gold
complex to assess nonspecific adsorption of a protein-gold complex
to
the tissue sections; (iii) incubation of the tissue sections
with the
enzyme-gold complex under nonoptimal conditions for biological
activity; and (iv) incubation of the sections with colloidal gold
alone
to assess nonspecific adsorption of the gold
particles.
Detection of cellulase activity.
We filled petri dishes with
PDA amended with 0.5% (wt/vol) low-viscosity CMC (Sigma, St. Louis,
Mo.) in 50 mM sodium acetate buffer (pH 5.0). Cellulysin, a
cellulolytic complex from Trichoderma viride
(Calbiochem-Novabiochem, La Jolla, Calif.), was used as a positive
control at a concentration of 1 mg/ml in the same buffer. Petri dishes
were divided into four sections, and drops (20 µl) of either
cellulysin, P. oligandrum supernatant, PDB alone, or sodium
acetate buffer (pH 5.0) were deposited on the CMC-enriched PDA. The
plates were incubated at 25°C in the dark for 24 h.
Enzymatically active areas were visualized with a 2UVTM
transilluminator (model IM 26E; UVP, San Gabriel, Calif.) operating at
365 nm after incubation of the plates with Calcofluor fluorescent
brightener 28 (Sigma). The amounts of cellulolytic activity were
estimated by the sizes of the enzymatically digested areas.
Cellulolytic effect of culture supernatant on P. parasitica.
Disks (2 mm3) of P. parasitica
growing on PDA were placed on microscope slides, covered with a thin
layer of 2% (wt/vol) water agar, and allowed to grow overnight at
25°C in a moist chamber. One drop (10 µl) of either P. oligandrum culture supernatant, cellulysin, or sodium acetate
buffer was placed on the hyphal filaments emerging from each disk.
Inoculated slides were incubated for 2 to 6 h at 25°C in a moist
chamber prior to examination with a light microscope (Zeiss Canada
Ltd., North York, Ontario, Canada) equipped with differential
interference contrast (Nomarski). Mycelial samples also were taken from
the inoculated slides and processed for electron microscope
investigation as described above.
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RESULTS |
Mycelial interactions between P. oligandrum and
P. parasitica.
In petri dish dual cultures, the first
apparent contact between hyphae of the two fungi occurred 2 days after
inoculation. At that time, the P. oligandrum hyphae were
intertwined with those of P. parasitica. In the subsequent
days, the P. oligandrum mycelium continued to grow and to
colonize the agar. When transferred to fresh medium after 6 days of
coculture, P. parasitica hyphae did not grow.
Time course investigation of the cellular events involved in the
P. oligandrum-P. parasitica interaction. (i) Events
preceding host cell penetration.
After 1 day of coculture, the
hyphae of P. oligandrum could be recognized by the greater
electron density of their protoplasm, and they encircled and/or were
closely appressed against hyphae of P. parasitica (Fig.
1a). The host cells were not obviously altered, although some slight cytoplasmic disorganization, primarily associated with increased vacuolation, was noticed. After 2 days of
dual culture, host cell disorganization increased, as shown by the
frequent retraction of the plasma membrane, which is usually correlated
with an early stage of cytoplasmic aggregation (Fig. 1b). Plasma
membrane retraction often was accompanied by deposition of a
fibrillo-granular network in the paramural space (Fig. 1b). These wall
appositions were heterogeneous in size, shape, and texture and could
appear either as small hemispherical protuberances or as elongated
deposits on a large portion of the host cell wall.
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FIG. 1.
Transmission electron micrographs of mycelial samples,
collected after 1 and 2 days of coculture of P. parasitica
(Ph) and P. oligandrum (Po), in the region where the two
fungi are interacting. (a) Hypha of P. parasitica encircled
by hyphae of P. oligandrum. Structural changes are mainly
characterized by an increase in the number of vacuoles (Va). Bar, 2 µm. (b) After 2 days, host cell disorganization is characterized by
local retraction of the plasma membrane from the cell wall and by an
early stage of cytoplasm (Cy) aggregation. A heterogeneous wall
apposition (WA) is formed in the paramural space. Bar, 2 µm.
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After 3 days of coculture, the extent and magnitude of the host
cellular changes increased (Fig.
2b through
d). Retraction
of the plasma membrane was
complete, and the wall appositions
were significantly larger. Section
labeling with gold-complexed
exoglucanase resulted in massive
deposition of gold particles
in the host cell wall and the wall
appositions (Fig.
2b and c).
The amount of wall-bound labeling appeared
to be 10-fold greater
than that observed for
Phytophthora
hyphae grown in single culture
(Fig.
2a). Specifically labeled,
electron-opaque vesicles, frequently
seen in the condensed cytoplasm
(Fig.
2b), could be enclosed in
invaginations of the plasma membrane,
where they apparently released
their labeled contents (Fig.
2c).
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FIG. 2.
Transmission electron micrographs of mycelial samples,
collected after 3 to 4 days of coculture of P. parasitica
(Ph) and P. oligandrum (Po), in the region where the two
fungi are interacting. Cellulosic beta-1,4-glucans are labeled with the
exoglucanase-gold complex. (a) P. parasitica grown in single
culture. The cell wall (CW) is specifically labeled, while the
cytoplasm and the organelles, including mitochondria (M) and vacuoles
(Va), are not labeled. Bar, 0.375 µm. (b and c) After 3 days, the
host structural changes include complete retraction of the plasma
membrane, condensation of the cytoplasm (Cy) and enlargement of the
wall apposition (WA). Gold labeling occurs in the host cell wall, the
wall appositions, and the electron-opaque vesicles (panel b, arrows),
which can also be enclosed in invaginations of the plasma membrane
(panel c, arrows). Bars, 0.75 µm. (d) After 4 days of coculture, the
space between the thickened cell wall (CW) and the small aggregated
cytoplasmic remnants (Cy) is filled with a material forming a tight
network (large arrow). Osmiophilic inclusions are embedded in the
network, giving the Phytophthora cell a honeycomb
appearance. Bar, 0.75 µm.
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By 4 days, the space between the thickened cell wall and the small
aggregated cytoplasmic remnants could be filled with a
heterogeneous
material forming a tight network (Fig.
2d) surrounded
by distorted
wall-like strands. Osmiophilic inclusions were embedded
in this
network, giving the entire system a honeycomb appearance
(Fig.
2d).
(ii) Host cell binding and penetration.
After 3 to 4 days of
coculture, the hyphae of P. oligandrum were tightly bound to
those of P. parasitica (Fig. 3a and
b). Cell walls of both fungi appeared to
be diffuse when they were in close contact (Fig. 3b), and it often was
difficult to distinguish them, although the thin cell wall of P. oligandrum usually was more electron dense than the thickened wall
of P. parasitica (Fig. 3b). Adhesion of P. oligandrum hyphae was usually accompanied by little wall
displacement (Fig. 3a). Host cell reactions, including the formation of
wall appositions, also were detected at sites of potential penetration
(Fig. 3a). Firm binding of P. oligandrum to its host
preceded host cell wall degradation and host penetration (Fig. 3c and
d). The lytic activity of P. oligandrum was confirmed by the
degradation of the thickened host cell wall and the enlarged wall
appositions (Fig. 4a and b). Lysis zones
were always free of exoglucanase-gold labeling (Fig. 4b).
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FIG. 3.
Transmission electron micrographs of mycelial samples,
collected after 3 to 4 days of coculture of P. parasitica
(Ph) and P. oligandrum (Po), in the region where the two
fungi are interacting. Cellulosic beta-1,4-glucans are labeled with the
exoglucanase-gold complex. (a and b) Hyphae of P. oligandrum
establish tight binding with the host cells (arrows). Adhesion of
P. oligandrum hyphae to the host cells is accompanied by
little wall displacement (panel a, arrow). Wall thickening occurs at
sites of potential antagonist penetration (panel a, double arrows). CW,
cell wall. Bars, 0.75 µm. (c and d) Firm binding of P. oligandrum to its host is associated with local cell wall
degradation (arrows). (c) Bar, 1.5 µm; (d) bar, 0.75 µm.
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FIG. 4.
Transmission electron micrographs of mycelial samples,
collected after 3 to 4 days of coculture of P. parasitica
(Ph) and P. oligandrum (Po), in the region where the two
fungi are interacting. (a and b) Lytic zones are free of
exoglucanase-gold labeling (arrows). (a) WA, wall apposition. Bar, 1.5 µm. (b) Bar, 0.75 µm. (c and d) Host cell penetration is achieved
by means of constricted hyphae of the antagonist (panel d, arrow).
There is a decrease in gold labeling some distance from the fungal
pathway (panel d, arrowhead). (c) Bar, 1.5 µm; (d) bar, 0.75 µm.
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Constricted hyphae of
P. oligandrum penetrated the host
cells (Fig.
4c and d). Channels of penetration often were much narrower
than the average hyphal diameter and usually were associated with
little wall displacement (Fig.
4d). Gold labeling decreased not
only
along the fungal antagonist pathway but also at some distance
from it
(Fig.
4d).
P. oligandrum ingress into the pathogen protoplasm coincided
with extensive cell alterations leading to complete dissolution
of the
host cytoplasm (Fig.
5a). At this stage,
P. parasitica hyphae appeared to be little more than empty
shells (Fig.
5a and
d). Cells of
P. oligandrum grew
abundantly in the host hyphae
and invaded the area that was originally
occupied by the host
cytoplasm (Fig.
5a and b). This colonization was
usually accompanied
by a generalized lytic activity that resulted in
host wall alterations
(Fig.
5b). The release of fibrillar fragments,
which were specifically
labeled by the gold-complexed exoglucanase,
occurred in areas
of host cell wall degradation (Fig.
5c). Extensive
digestion of
the host cell walls resulted in cell perforation in many
places
(Fig.
5d).
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FIG. 5.
Transmission electron micrographs of mycelial samples,
collected after 4 to 5 days of coculture of P. parasitica
(Ph) and P. oligandrum (Po), in the region where the two
fungi are interacting. (a through c) Cells of the antagonist
proliferate in the host hyphae, resulting in host wall alterations
(panels a and b, arrows). Labeled wall fragments are released (panel c,
arrowheads). (a and b) Bars, 1.5 µm; (c) bar, 0.75 µm. (d) Host
cell is perforated in many places (arrow). Bar, 1.5 µm.
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Eventually, cells of
P. oligandrum multiplied so extensively
in the host hyphae that it was difficult to identify free space
in the
area that was originally occupied by the host cytoplasm
(Fig.
6). Under such pressure, the
P. parasitica hyphae apparently
burst, leaving only some tiny wall
fragments to indicate the former
presence of
Phytophthora
cells (Fig.
6).
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FIG. 6.
Transmission electron micrographs of mycelial samples,
collected after 4 to 5 days of coculture of P. parasitica
(Ph) and P. oligandrum (Po), in the region where the two
fungi are interacting. Active multiplication of the antagonist results
in apparent bursting of the host hyphae (panel a, large arrow) and in
release of the actively multiplying P. oligandrum hyphae.
Bars, 2 µm.
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All control tests, including previous adsorption of the enzyme-gold
complexes with their corresponding substrate molecules,
yielded
negative results (data not
shown).
Cellulase activity in the culture supernatant of P. oligandrum.
The culture supernatant of P. oligandrum
was applied to PDA plates amended with the substrate CMC, and its
activity was compared to that of a pure cellulolytic complex from
T. viride. Following calcofluor staining of the cellulosic
polymer, negative zones, corresponding to the areas where drops were
deposited, were easily seen for both the cellulysin from T. viride and the culture supernatant of P. oligandrum
(data not shown). By contrast, no signal was obtained with either PDB
or sodium acetate buffer alone (data not shown).
We observed mycelium of
P. parasitica growing in water agar
on microscope slides at 1-h intervals. Mycelia exposed to either
PDB or
sodium acetate buffer had hyphae delimited by a thin cell
wall that
contained dense polyribosome-enriched cytoplasm in which
a large number
of organelles, including mitochondria and small
vacuoles, were found
(Fig.
7a). Incubation of sections with
the
beta-1,4-exoglucanase-gold complex resulted in regular deposition
of gold particles over the cell wall, with some preferential labeling
of the internal wall (Fig.
7a).
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FIG. 7.
Transmission electron micrographs of mycelial samples
collected from agar-coated slides 2 to 6 h after inoculation of
P. parasitica (Ph): cellulolytic effect of P. oligandrum (Po) culture supernatant on P. parasitica
hyphae. (a) P. parasitica exposed to PDB (control). Hyphae
are delimited by a regularly labeled cell wall (CW) and contain a dense
cytoplasm (Cy) in which mitochondria (M) and vacuoles (Va) are visible.
Bar, 0.25 µm. (b) After 2 h of exposure to the culture
supernatant of P. oligandrum, local retraction of the plasma
membrane is accompanied by formation of wall appositions (WA). Bar, 0.5 µm. (c) After 3 h of exposure, complete retraction of the plasma
membrane (PM), condensation of the cytoplasm, and loss of cell wall
rigidity are detected. Bar, 0.25 µm. (d through g) Upon prolonged
exposure (4 h or more), a large number of unlabeled areas are detected
in the outermost wall layers of Phytophthora hyphae (panel
d, arrowheads). These areas can extend to large portions of the cell
wall (panel e, arrow) in which labeling is restricted to small wall
fragments (panel f, arrowheads). Alteration of the wall appositions
(panel g, large arrow) leads to release of labeled fragments (panel g,
arrowheads). (d) Bar, 1 µm; (e) bar, 0.5 µm; (f) bar, 0.25 µm;
(g) bar, 0.5 µm. (h) After 6 h of exposure,
Phytophthora hyphae are surrounded by a slightly labeled
cell wall. Bar, 0.5 µm.
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Mycelia exposed to
P. oligandrum culture supernatant had
morphological and structural alterations that could be detected as
soon
as 2 h after exposure. These changes, primarily local retraction
of the plasma membrane and the formation of wall appositions,
usually
were restricted to well-delineated wall areas (Fig.
7b).
By 3 h
after exposure, the plasma membrane was completely retracted,
the
cytoplasm had condensed, and the cell wall was no longer rigid
(Fig.
7c).
Following 4 to 5 h of exposure to the culture supernatant, most
P. parasitica cells were severely damaged (Fig.
7c through
g). When cells were incubated with the gold-complexed exoglucanase,
numerous unlabeled areas were seen in the outermost wall layers
of
Phytophthora hyphae (Fig.
7d). These areas often included
large
portions of the cell wall (Fig.
7e) in which labeling was
restricted
to relatively small wall fragments (Fig.
7f). In some cases,
the
wall appositions also were altered and labeled fragments were
released (Fig.
7g).
By 6 h after exposure to the culture filtrate, most
Phytophthora hyphae (~80%) were surrounded by a thin,
wavy cell wall which
was labeled by a few scattered gold particles
(Fig.
7h).
Changes similar to those induced by the culture supernatant of
P. oligandrum also were observed after exposure to cellulysin
from
T. viride (data not shown). However, the alterations in
response
to cellulysin occurred earlier (within 2 h) and were more
severe
than those induced by the
P. oligandrum culture
filtrate.
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DISCUSSION |
In this study, we showed that the oomycete fungus P. parasitica is highly vulnerable to attack by the antagonistic
fungus P. oligandrum. Our results provide ultrastructural
evidence that P. oligandrum-mediated antagonism is a
multifaceted process that requires the synergistic contribution of
several mechanisms, including cell surface attachment and production of
hydrolytic enzymes (e.g., cellulases). According to our observations,
the process of Phytophthora colonization by P. oligandrum 1010 involves a chronological sequence of events,
including (i) attachment and local penetration of the antagonist into
the pathogen hyphae, (ii) induction of a host structural response,
(iii) alteration of the host protoplasm, and (iv) active multiplication
of the antagonistic cells in the pathogen hyphae, leading to host cell breakdown.
One of the earliest events of the antagonistic process was the apparent
affinity of P. oligandrum hyphae for cells of the pathogenic
fungus (Fig. 1a). The antagonist is attracted to the host cells by an
unknown mechanism that probably involves specific chemical stimuli
(32) or chemotropic growth (11). Support for the
hypothesis that molecular signals are exchanged between the fungi
includes the host cell changes initiated prior to adhesion of P. oligandrum hyphae and the similarities between these reactions and
those seen in other fungal cells exposed to antibiotics and/or fungicides (3, 19, 23). In particular, alterations in
membrane permeability could result in internal osmotic imbalances
(25), leading to cytoplasm disorganization and aggregation
such as that which precedes parasitism and subsequent internal
colonization of P. parasitica cells.
Host cell changes were correlated with abnormal deposition of a
cellulose-enriched material between the invaginated plasma membrane and
the host cell wall. We think that the deposits are a defense reaction
to restrict ingress of the antagonist and to shield the cell wall from
hydrolytic enzymes and toxins. The formation of structural barriers as
a response by resistant plant cultivars to fungal attack (4, 8,
16) is well documented, but relatively little is known about
defense-related structural responses in fungi. There are at least two
hypotheses that could explain the response of the
Phytophthora cells. First, the production of low levels of
hydrolytic enzymes, e.g., beta-1,3-glucanases, by P. oligandrum may have allowed the release of elicitor-active
molecules, e.g., beta-1,3-glucans, from the host cell wall that
ultimately increased the activity of cellulose synthase. In plants,
glucan preparations derived from fungal cell walls can stimulate a
resistance response (14). Alternatively, the signal molecule
could be a toxin or an antibiotic secreted by the antagonist.
Alteration of the lipid composition of the plasma membrane of
Phytophthora hyphae, possibly resulting from the binding of
such molecules, may have induced deregulation of membrane-bound
enzymes, resulting in areas of abnormal wall-like deposition.
Whatever the origin and role of the deposited material, the antagonist
can penetrate this barrier by altering its structure (Fig. 5).
Degradation of the host cell wall and the wall appositions always was
preceded by firm binding of P. oligandrum to its host. Cell
surface molecules play an important role in cell-to-cell interactions
in many biological systems (22, 29), and early recognition
events, mediated by molecules with sugar-binding affinity, are known to
be important determinants in establishing the mycoparasitic relationship between Trichoderma spp. and their target hosts
(2, 6). We think that the tight binding observed between
hyphae of the antagonist and cells of P. parasitica is
mediated by a specific cell surface recognition process which, in turn,
triggers a series of events that includes host wall penetration and
cell invasion.
The successful penetration of the thickened host cell wall and the
enlarged wall appositions by P. oligandrum hyphae suggests that large amounts of cellulolytic enzymes were produced. The labeling
pattern of cellulose showed that the integrity of this compound was
affected in wall areas adjacent to P. oligandrum cells and
also at a distance from the sites of antagonist entry, suggesting that
cellulases may have diffused extracellularly. Production of
extracellular lytic enzymes, e.g., beta-1,3-glucanases, lipases, and
proteases, by P. oligandrum may be involved in antagonism against an array of pathogenic fungi (17, 24, 26, 27). Our
results show that at least under our experimental conditions, P. oligandrum could produce large amounts of cellulases in
substrate-free liquid medium and that these enzymes were nearly as
effective as the cellulolytic complex from T. viride in
degrading both CMC and Phytophthora wall-bound cellulose.
In vitro demonstration that cellulases produced by P. oligandrum may play a major role in biological control of P. parasitica provides an incentive to develop this organism and
these enzymes as a biological control agent for commercial use.
However, the most important problem to be solved prior to large-scale
application of biocontrol strains is the ability to predict the
behavior of these strains in the field based on laboratory results. The
possibility of improving and maintaining the biocontrol activities of
fungal antagonists by genetic manipulation techniques is promising.
Future research should focus on development of transgenic P. oligandrum strains capable of producing large quantities of
cellulases while the intrinsic vigor and the ecological competence of
the fungus are preserved. Manipulated biocontrol fungi need to become
more predictable and reliable for use in the field and could
potentially reduce the quantity of fungicides required to produce
disease-free plants.
| |
ACKNOWLEDGMENTS |
We thank J. Hockenhull (The Royal Veterinary and Agricultural
University, Copenhagen, Denmark) and M. Ponchet (INRA, Antibes, France)
for providing the isolates of P. oligandrum and P. parasitica, respectively, and C. Garand, A. Goulet, and H. Chamberland (Laval University, Québec, Canada) for technical assistance.
This work was supported by grants from the Fonds Québécois
pour la Formation de Chercheurs et l'Aide à la Recherche (FCAR), the Natural Sciences and Engineering Council of Canada (NSERC), the
GIS-LBIO Program (ONIFLHOR), and the Brittany Regional Council (France).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Recherche en
sciences de la vie et de la santé, Pavillon C.E. Marchand,
Université Laval, Sainte-Foy, Québec, Canada G1K 7P4.
Phone: (418) 656-7517. Fax: (418) 656-7176. E-mail:
nben{at}rsvs.ulaval.ca.
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Abstract
Introduction
