![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Applied and Environmental Microbiology, March 1999, p. 1061-1070, Vol. 65, No. 3
0099-2240/99
Induction of Defense Responses in Cucumber Plants
(Cucumis sativus L.) by the Biocontrol Agent
Trichoderma harzianum
I.
Yedidia,1
N.
Benhamou,2 and
I.
Chet1,*
Department of Plant Pathology and
Microbiology, Faculty of Agriculture, The Hebrew University of
Jerusalem, Rehovot 76100, Israel,1 and
Recherche en Sciences de la Vie et de la Sante, Laval
University, University City, Quebec G1K 7P4,
Canada2
Received 12 August 1998/Accepted 14 December 1998
 |
ABSTRACT |
The potential of the biocontrol agent Trichoderma
harzianum T-203 to trigger plant defense responses was
investigated by inoculating roots of cucumber seedlings with
Trichoderma in an aseptic, hydroponic system.
Trichoderma-treated plants were more developed than
nontreated plants throughout the experiment. Electron microscopy of
ultrathin sections from Trichoderma-treated roots revealed
penetration of Trichoderma into the roots, restricted
mainly to the epidermis and outer cortex. Strengthening of the
epidermal and cortical cell walls was observed, as was the deposition
of newly formed barriers. These typical host reactions were found
beyond the sites of potential fungal penetration. Wall appositions
contained large amounts of callose and infiltrations of cellulose. The
wall-bound chitin in Trichoderma hyphae was preserved, even
when the hyphae had undergone substantial disorganization. Biochemical
analyses revealed that inoculation with Trichoderma
initiated increased peroxidase and chitinase activities within 48 and
72 h, respectively. These results were observed for both the roots
and the leaves of treated seedlings, providing evidence that T. harzianum may induce systemic resistance mechanisms in cucumber plants.
| |
INTRODUCTION |
In the past few years,
Trichoderma spp., the most common saprophytic fungi in the
rhizosphere, have received considerable attention as potential
biocontrol agents for a number of soilborne pathogens (9,
35). The mechanisms by which Trichoderma isolates control pathogenic populations in the rhizosphere have been extensively studied. Recent progress in the purification and identification of
Trichoderma metabolites has led to the notion that, in most cases, the antagonistic process relies on the production of antibiotics (16) and/or hydrolytic enzymes (17, 18, 20)
associated with possible competition for nutrients in the rhizosphere
(37). In spite of the increasing amount of research devoted
to the antimicrobial activity of Trichoderma spp. in vitro
(19, 23), knowledge of the exact mechanisms responsible for
the observed reduction of disease incidence following soil treatment
with Trichoderma propagules is still incomplete. Indeed,
most studies have focused on microbial interactions and not on the
possible involvement of the host plant, although circumstantial
evidence correlating increased plant growth response with penetration
of Trichoderma harzianum into the root system has been
reported (25).
In recent studies, substrate amendment with T. harzianum
resulted in enhanced plant growth throughout the growing season
(1, 19). Biocontrol of minor pathogens in the rhizosphere,
improved mineral uptake, nutrient release from the soil and organic
matter, and enhanced plant hormone production (6, 19, 22)
may partially explain the Trichoderma-mediated enhanced
growth response. However, the possibility that Trichoderma
spp. interact with root tissues and induce host plant resistance to
pathogens has seldom been raised. This latter concept, along with the
recent demonstration that infection with beneficial fungi, such as
endomycorrhizal fungi, causes host plants to respond more rapidly and
efficiently to pathogen attack (12, 29), raises the
following question: to what extent might nonpathogenic fungi, such as
Trichoderma spp., stimulate the plant defense response,
leading to the activation of genes and ultimately to the accumulation
of defense molecules?
A growing body of evidence from various studies indicates that
increased resistance of arbuscular mycorrhizal roots may be associated
in part with marked metabolic changes in the host, including enhanced
production of peroxidases and phenolic compounds (39);
accumulation of hydrolases, such as chitinases and 
-1,3-glucanases, with antimicrobial potential (12, 38); and deposition of
structural polymers, such as lignin (7) and
hydroxyproline-rich glycoproteins (2). If one considers that
the increased production of peroxidases and phenolic compounds may be
of key importance in the resistance process (10) and that
the accumulation of structural substances may increase the mechanical
strength of the host cell walls, the induction of such defense
mechanisms by beneficial fungi would likely inhibit or at least
restrict pathogen invasion. However, at present, the situation is not
clearly defined, and additional research is needed to confirm the
effective stimulation of the plant defense system upon infection by
nonpathogenic fungi. Nevertheless, these earlier observations, together
with the finding that Trichoderma spp. are able to promote
plant growth (22), raise key questions. (i) Does
Trichoderma penetrate the root tissues, and if so, what are
the pattern of fungal colonization and the relationship with the host
plant? (ii) Is Trichoderma capable of stimulating the plant
to defend itself through the accumulation of defense molecules?
In an attempt to answer these important questions, the objectives of
the research described here were (i) to determine whether or not
T. harzianum penetrates the epidermis of cucumber roots and
develops in the inner tissues, (ii) to delineate the biological events
associated with the cucumber-Trichoderma interaction, and (iii) to biochemically investigate the induction of some potential defense molecules. As a result, we present the first conclusive evidence that T. harzianum penetrates and grows in the
epidermis and outer cortex and stimulates the plant defense system,
leading to the production of biochemical and structural compounds.
| |
MATERIALS AND METHODS |
Plant material.
Seeds of cucumber (Cucumis
sativus L. cv. Delila) from Gedera Seeds, Gedera, Israel, were
used in this experiment. Plant growth medium (PGM) consisted of, per
liter, 0.24 g of MgSO4, 0.04 g of
K2HPO4, 0.17 g of
K2SO4, 0.344 g of CaSO4 · H2O, 0.64 g of NH4NO3, and 1 ml of the following trace elements (per liter of stock solution):
0.05 g of FeCl3, 0.728 g of KCl, 1.546 g of
H3BO3, 0.846 g of MnSO4 · H2O, 0.375 g of ZnSO4 · 7H2O, 0.125 g of CuSO4 · 5H2O, 0.081 g of H2MoO4, and 0.001 g of CoCl2 · 6H2O (pH 7.0).
Fungal material.
T. harzianum T-203 (13)
was grown on potato dextrose agar (Difco). Synthetic medium for
T. harzianum consisted of, per liter, 15 g of glucose,
0.2 g of MgSO4 · 7H2O, 0.6 g
of K2HPO4, 0.15 g of KCl, 1 g of
NH4NO3, and 1 ml of the following trace
elements (per liter of stock solution: 0.005 g of
FeSO4 · 7H2O, 0.006 g of
MnSO4 · H2O, 0.004 g of
ZnSO4 · H2O, and 0.002 g of
CoCl2 (31). One milliliter (109 to
1010 spores, as counted with a hemocytometer) of 7-day-old
T. harzianum cultured on potato dextrose agar was used as
the inoculum for a 250-ml flask containing 100 ml of synthetic medium.
The flask was shaken at 150 rpm for 24 h at 30°C to allow spore
germination. After 24 h, the mycelial inoculum was separated from
the growth medium by centrifugation at 10,000 × g and
4°C and washed twice in 100 ml of distilled water.
Axenic growth system.
Growth chambers were made of
bicomponent, autoclavable, transparent polycarbonate boxes (Biological
Industries, Beit Haemek, Israel). The top part was 13.7 cm high (10.7 by 10.7 cm), and the bottom part was 5.2 cm high (10.7 by 10.7 cm),
holding up to 500 ml. Seeds were surface disinfected in 70% ethanol
for 2 min, followed by 2.0% NaOCl for 2 min, and thoroughly washed
with sterile distilled water. Seeds (24 per box) were placed on a
sterile gauze sheet over a sterile stainless steel screen, which held them 1 cm above 300 ml of PGM. Plants were grown in a controlled environment: 26°C, 80% relative humidity, and a circadian cycle of
14 h of light and 10 h of dark. A Tygon autoclavable tube was inserted into the bottom part through a breach and hooked to an oxygen
stone to aerate the liquid medium with atmospheric air filtered through
a 0.45-µm-pore-size filter.
Plant inoculation with T. harzianum.
Mycelial inoculum
was added under aseptic conditions to the PGM of 7-day-old seedlings to
a final concentration of ±105 germinated spores/ml. The
concentration of the Trichoderma inoculum was determined to
be 10
3 times the concentration used for biocontrol under
greenhouse conditions (107 to 108 CFU/g of
soil). Thus, ±105 germinated spores/ml of PGM were used.
Control plants were treated with sterile distilled water.
Tissue processing for electron microscopic studies.
Root
samples (2 mm3) collected from the crown and the main root
at sites of attempted fungal penetration 5 days after inoculation were
embedded in 1% (wt/vol) aqueous Bacto Agar prior to fixation by
immersion in 3% (vol/vol) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h at room temperature. Root samples were postfixed in 1% (wt/vol) osmium tetroxide in the same buffer for 1 h at 4°C, dehydrated in a graded ethanol series, and embedded in Epon 812. Ultrathin sections (0.1 µm) were cut with a diamond knife and collected on Formvar-coated nickel grids. The sections were
either treated with uranyl acetate and lead citrate for immediate examination in a JEOL 1200 EX transmission electron microscope operating at 80 kV or further processed for cytochemical labeling. For
each treatment, an average of five samples from five different roots
were investigated. For each sample, 10 to 15 ultrathin sections were examined.
Preparation of the gold-complexed probes.
Colloidal gold
with particles averaging 12 nm in diameter was prepared as described by
Frens (14) with sodium citrate as the reducing agent. To
localize cellulosic compounds, a -1,4-exoglucanase (-1,4-glucan
cellobiohydrolase) purified from a cellulase produced by T. harzianum was directly complexed to colloidal gold at pH 9.0 (5). Callose, a polymer of -1,3-glucans, was localized with -1,3-glucanase extracted and purified from tobacco plants reacting hypersensitively to tobacco mosaic virus (24). The enzyme was complexed to gold particles at pH 5.5 (4).
N-Acetylglucosamine residues (chitin) were localized by a
two-step procedure (3) with wheat germ agglutinin (WGA) as
the first-step reagent and gold-complexed ovomucoid as the second-step reagent. The ovomucoid was complexed to the gold at pH 5.4. All gold-conjugated probes were stored at 4°C and centrifuged at 2,600 × g for 2 min before use.
Cytochemical labeling.
Labeling with the gold-complexed
-1,4-exoglucanase and -1,3-glucanase was performed by first
incubating the ultrathin root sections for 5 to 10 min in 1 drop of
0.01 M sodium phosphate-buffered saline (PBS) containing 0.02%
(wt/vol) polyethylene glycol 20000 at pH 6.0, corresponding to the
optimal activity of the enzyme. Sections were transferred to 1 drop of
gold-complexed probe for 30 to 60 min at room temperature in a moist
chamber. After careful washing with PBS (pH 7.2) and rinsing with
distilled water, sections were treated with uranyl acetate and lead
citrate and observed in the JEOL 1200 EX transmission electron microscope.
For the indirect labeling of chitin, sections were first incubated in 1 drop of PBS (pH 7.2), transferred to 1 drop of WGA diluted 1:60 in PBS
(pH 7.2), and finally incubated in 1 drop of ovomucoid-gold complex
(3). Sections were treated as already described. The
specificity of the different labeling procedures was assessed by the
following control tests: (i) addition of the corresponding substrate to
each gold-complexed probe for a competition experiment (-1,4-glucans
from barley [1 mg/ml] for the -1,4-exoglucanase-gold complex,
laminarin [1 mg/ml] for the -1,3-glucanase-gold complex, and
N-N'-N"-triacetylchitotriose [1 mg/ml] for WGA; (ii)
substitution of the gold complex under study with a bovine serum
albumin-gold complex to assess nonspecific adsorption of the
protein-gold complex to the tissue sections; (iii) incubation of the
tissue sections with the enzyme-gold complexes under nonoptimal
conditions for biological activity; and (iv) incubation of the tissue
sections with colloidal gold alone to assess nonspecific adsorption of the gold particles to the tissue sections.
Protein extraction.
Seedlings were divided into portions,
stems were excluded, and roots and leaves were separated, washed under
running tap water for 5 min, dried gently, weighed, and ground with a
mortar and pestle under liquid nitrogen. The ground matter was
homogenized (2 min, 4°C) in phosphate buffer (1:2 w/v, pH 6, 0.05 M)
by use of Corex tubes and an ULTRA-TURRAX apparatus (TP 18/10;
IKA-WERK, Staufen, Germany). The homogenate was centrifuged twice at
10,000 × g and 4°C, and the supernatant was
collected and kept at 
20°C.
Detection of chitinase and peroxidase activities.
The total
chitinase activity assay was based on the colorimetric determination of
p-nitrophenyl cleaved from a chitin-analogous substrate,
p-nitrophenyl--D-N,N'-diacetylchitobiose
(PNP) (20, 33). A crude enzyme preparation and 10 µl of
PNP stock solution (2 mg/ml) were added to 50 mM acetate buffer (pH
5.0) to a total volume of 0.5 ml and incubated for 2 h in a water
bath at 37°C. The reaction was terminated with 0.5 ml of 0.2 M
Na2CO3. An extinction coefficient of 7 × 103 mM1 cm1 at 410 nm was used
to determine p-nitrophenyl release from the substrate
(Varian Techtron DMS100 UV-visible spectrophotometer). Chitinase
activity was expressed as millimoles of PNP produced per gram of fresh
tissue per hour.
Peroxidase activity was assayed spectrophotometrically at 610 nm with
phenol red as a substrate. The complete reaction mixture (1 ml, 37°C)
contained 10 to 20 µl of a crude enzyme preparation, 50 µl of 0.2%
(wt/vol) phenol red, and 50 mM sodium citrate (pH 4.2). Reactions were
initiated with 10 µl of 1 mM hydrogen peroxide and stopped after 3 min with 40 µl of 2 N sodium hydroxide. The optical density was
detected at 610 nm as described above. The absorbance was recorded at
610 nm and calculated with a molar extinction coefficient of 22,000 M1 cm1 for the oxidized product
(34). Peroxidase activity was expressed as millimoles of
phenol red oxidized per gram of fresh tissue per minute.
Enzymatic assays consisted of 15 plants per treatment and were repeated
in at least four independent experiments, which showed similar results.
The average of two representative experiments is shown (see Fig. 5 and
6).
| |
RESULTS |
Cytology of infection of cucumber root tissues by T. harzianum.
A hydroponic growth system was developed to control
growth conditions and medium composition and to eliminate unwanted
microorganisms. The growth chambers allowed the development of cucumber
seedlings for up to 21 days. An increased growth response was observed
during the growth period in the Trichoderma-treated plants.
This experimental approach confirms, for the first time, the direct
impact of the Trichoderma fungus on treated plants. Visual
monitoring of the progression of the fungal-plant interaction indicated
complete attachment of the fungal hyphae to the root surface as early
as 48 h postinoculation. Transverse sections of root samples taken from cucumber seedlings grown in the presence of T. harzianum revealed a large number of fungal hyphae growing on the
root surface and establishing intimate contact with the host exodermis
(Fig. 1A and B). About 30 root sections
were observed, providing evidence that the fungus penetrates the root
epidermis without extensive host cell wall degradation (Fig. 1B). The
fungus then multiplies in the epidermis and progresses toward the
cortical area, mainly by intercellular growth (Fig. 1A, arrows). Fungal
ingress into the innermost root tissues was seldom observed, except in
some localized areas, where Trichoderma cells could be seen
in the endodermis (Fig. 1C).
View larger version (109K):
[in this window]
[in a new window]
|
FIG. 1.
Transmission electron micrographs of T. harzianum-inoculated cucumber root tissues. A large number of
fungal hyphae (T) develop at the root surface. Trichoderma
hyphae penetrate the root epidermis (Ep) and progress toward the
cortical area (CA), mainly by intercellular growth (arrows in A). Wall
appositions (WA) are seen in noninvaded host cells beneath the
colonized areas. Fungal colonization of the epidermis and cortex is not
associated with host cell alterations or cell wall digestion (C). IS,
intercellular spaces; VS, vascular stele. Bars: A, 10 µm; B, 1.5 µm; C, 5 µm.
|
|
Fungal colonization of the epidermis and cortex usually involved only a
few hyphae, characterized by the high electron density of their
cytoplasm, and was not associated with the host cell alterations or
cell wall digestion reported for various other host-pathogen
interactions (Fig. 1A and C).
A closer examination of the invaded areas revealed, however, the
occurrence of host reactions at sites of attempted fungal penetration.
One of the most striking of these was the formation of heterogeneous
wall appositions in the noninfected host cells adjacent to invaded
cells (Fig. 2A and B). These appositions
varied in size and shape, ranging from small, hemispherical or
dome-like protuberances (Fig. 2A) to elongated deposits along a large
portion of the host cell wall (Fig. 2B). The extensive heterogeneity of the newly formed wall appositions was clearly demonstrated in some
areas, where the material accumulating at the junction of adjacent host
cells appeared ultrastructurally different in terms of arrangement
(Fig. 2B). The deposited material was either stratified and bordered by
a layer of aggregated material (Fig. 2B, large arrow) or granular and
of very high electron density (Fig. 2B, small arrow). Frequently, the
appositions were delimited by a band of osmiophilic material that
released small droplets into the cell lumen (Fig. 2A, arrowheads).
Whether this densely stained material originated from an aggregation of
the host cytoplasm or was newly synthesized as a response to infection
remains unclear.
View larger version (153K):
[in this window]
[in a new window]
|
FIG. 2.
Transmission electron micrographs of T. harzianum-inoculated cucumber root tissues. (A and B)
Heterogeneous wall appositions (WA) are formed in noninfected host
cells adjacent to invaded cells. The material accumulating at the
junction of adjacent host cells was either stratified and bordered by a
layer of aggregated material (large arrow in B) or granular and of very
high electron density (small arrow in B). Frequently, the appositions
were delimited by a band of osmiophilic material that released small
droplets into the cell lumen (arrowheads in A). (C and D) Unsuccessful
attempts by the fungus to penetrate WA are observed.
Trichoderma hyphae (T) penetrating such appositions show
marked signs of alteration characterized by morphological changes and
even necrosis of the penetration peg. IS, intercellular spaces. Bars: A
and D, 1 µm; B, 0.5 µm; C, 0.25 µm.
|
|
The host cell wall itself displayed a higher electron density than
controls, strongly suggesting infiltration of structural molecules
(Fig. 2A). Unsuccessful attempts of fungi to penetrate wall appositions
were frequently recorded (Fig. 2C and D). Hyphae penetrating such
appositions showed marked signs of alteration, characterized by
morphological changes and even necrosis of the penetration peg (Fig. 2C
and D).
As expected, application of the -1,4-exoglucanase-gold complex to
sections of colonized cucumber root tissues resulted in the heavy
deposition of gold particles over the host cell walls (Fig.
3A). In contrast, labeling was nearly
absent over the host cytoplasm and organelles. Labeling also occurred
over the wall appositions but was irregular and usually involved only a
few randomly distributed gold particles (Fig. 3A). Interestingly, Trichoderma hyphae displayed the ability to locally disrupt
cellulose-enriched host cell walls (Fig. 3A, arrows), although they
appeared to be halted by the wall appositions. Control tests, including
preincubation of the enzyme-gold complex with -1,4-glucans prior to
section labeling, resulted in the absence of labeling over both cell
walls and wall appositions (data not shown). Upon incubation of
sections with tobacco -1,3-glucanase, many gold particles were
detected over the wall appositions (Fig. 3B). A qualitative evaluation of the labeling clearly showed that young appositions, mainly characterized by their loose arrangement and their low density (Fig.
3B), were more intensely labeled than more mature ones, on which
osmiophilic flecks were deposited (Fig. 3C). In such cases, labeling
occurred preferentially over the outermost layer (Fig. 3C).
Electron-opaque vesicles, either enclosed in the wall appositions or
apparently free in the cytoplasm, were frequently seen (Fig. 3D,
arrows). Such vesicles were always labeled with a substantial number of
gold particles. Control tests, including incubation of the enzyme-gold
complex with laminarin prior to section labeling, yielded negative
results (data not shown).
View larger version (158K):
[in this window]
[in a new window]
|
FIG. 3.
Transmission electron micrographs of T. harzianum-inoculated cucumber root tissues. (A) Labeling with the
-1,4-exoglucanase-gold complex to localize cellulose. The host cell
walls (HCW) are heavily labeled. Labeling also occurs over the wall
appositions (WA) but is irregularly distributed. Trichoderma
hyphae (T) display the ability to locally disrupt the
cellulose-enriched HCW (arrows). Bar, 0.25 µm. (B to D) Labeling with
the tobacco -1,3-glucanase to localize callose. Young WA, mainly
characterized by their loose arrangement and their low density (B), are
more intensely labeled than the more mature ones, labeling of which
occurs preferentially over the outermost layer (C). Electron-opaque
vesicles, either enclosed in the WA (arrow pointing up in D) or
apparently free in the cytoplasm (arrows pointing down in D), are
labeled by a substantial number of gold particles. Bars, 0.25 µm.
|
|
Another prominent response to Trichoderma infection in
cucumber plants was the occlusion of most intercellular spaces in the epidermis and outer root cortex with a dense material that showed various degrees of compactness and electron opacity (Fig.
4A to C). Fungal cells trapped in this
material exhibited some morphological alterations (Fig. 4A) and were
apparently halted in their development (Fig. 4B). Incubation with
tobacco -1,3-glucanase did not result in substantial labeling of the
electron-opaque material, except next to the fungal cells, where a few
gold particles could be seen (Fig. 4C). The material was similarly not
labeled with the -1,4-exoglucanase-gold complex (data not shown).
View larger version (152K):
[in this window]
[in a new window]
|
FIG. 4.
Transmission electron micrographs of T. harzianum-inoculated cucumber root tissues. Most intercellular
spaces (IS) in the epidermis and outer root cortex are occluded with a
dense material (AM) that shows various degrees of compactness and
electron opacity. Fungal cells trapped in this material are
morphologically altered. Upon incubation with the tobacco
-1,3-glucanase, this material appears unlabeled, except next to the
fungal cells, where a few gold particles can be seen (C). An
osmiophilic material (OM) coating the host cell walls surrounds the
invading hyphae (arrows in D). Upon labeling with WGA-ovomucoid-gold
complex for the localization of chitin, regular deposition of gold
particles occurs over the coated fungal cell walls; arrows show the OM
coating (E). T, Trichoderma hyphae; WA, wall appositions.
Bars: A, D, and E, 0.5 µm; B and C, 1 µm.
|
|
In addition to the formation of wall appositions and the occlusion of
intercellular spaces at sites of attempted fungal penetration, other
host cell reactions included the deposition of an amorphous, osmiophilic material which coated the host cell walls and surrounded the invading hyphae by means of elongated strands of aggregated material (Fig. 4D and E, arrows). Sections labeled with the
WGA-ovomucoid-gold complex revealed a regular deposition of gold
particles over the coated fungal cell walls (Fig. 4E).
Activation of defense mechanisms.
Chitinase and peroxidase
activities are commonly expressed during plant-fungus interactions
(10, 21). These activities in roots and leaves were measured
at 24, 48, 72, and 120 h postinoculation. Chitinase activity
peaked at 72 h in both leaves and roots of treated plants (Fig.
5), while in nontreated plants, activity increased gradually with time. At peak chitinase activity, treated roots showed a 6.4-fold increase and treated leaves showed a 3.2-fold increase compared with nontreated plants. Peroxidase activity peaked at
48 h after inoculation in treated roots (Fig.
6), whereas it remained constant with
time in nontreated plants. In treated leaves, peroxidase activity
peaked at 72 h after inoculation, while nontreated plants showed a
gradual increase with time. From 72 to 120 h, a two- to threefold
decrease in both activities in the leaves and roots of treated plants
was observed.
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 5.
Chitinase activity in roots (A) and leaves (B) of
7-day-old cucumber seedlings inoculated with Trichoderma
( ) at 24 to 120 h and uninoculated controls ( ). Bars
represent 1 standard deviation.
|
|
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 6.
Peroxidase activity in roots (A) and leaves (B) of
7-day-old cucumber seedlings inoculated with Trichoderma
() at 24 to 120 h and uninoculated controls (). Bars
represent 1 standard deviation.
|
|
| |
DISCUSSION |
In recent years, interest in the ability of beneficial
microorganisms to induce resistance in plants has grown, particularly with respect to their use as environmentally safe controllers of plant
diseases (27, 29, 42). Among these microorganisms, saprophytic fungi have received only little attention as potential inducers of resistance (11). Nevertheless, saprophytic
isolates that induced resistance also promoted the growth of cucumber
plants (28, 30). In the present study, a hydroponic growth
system was used to inoculate the roots of a host plant with
monocultures of Trichoderma under predefined conditions.
This approach enabled us to visualize the progression of the
interaction to complete attachment of the fungal hyphae to the outer
root tissue of cucumber seedlings. The interaction was also expressed
in terms of biochemical and morphological changes induced in the
cucumber host by the fungus, among which the increased growth response
was the most salient, as described in earlier studies with soil
(1, 19, 22, 25). Several hypotheses, including the control
of minor pathogens (26), have been put forward to explain
the effects of Trichoderma on plants. The results of our
study in an aseptic environment with Trichoderma
monocultures suggest that a direct plant-fungus interaction is
responsible for the increased growth response as well as other
responses in the plant.
In soils, plant disease suppression by Trichoderma spp. used
as biocontrol agents has been widely documented (8, 9, 19,
37): it is considered to be a multifaceted process that requires
the synergistic contribution of several mechanisms, which may include
activation of the plant defense system (25). In the present
study, we examined the effect of a fungal biocontrol agent on plants.
The synthesis of pathogenesis-related (PR) proteins is one of the most
common defense mechanisms triggered in plants following infection with
inducing agents (10, 21, 40). Our work shows increased
production of PR proteins in cucumber seedlings following
Trichoderma application. Increased activities of chitinase and peroxidase were observed in both the roots and the leaves of
Trichoderma-treated plants relative to nontreated ones as
early as 48 h postinoculation. Moreover, the increase in enzyme
activities in the leaves suggests a systemic defense response to the
presence of Trichoderma in the rhizosphere. Strikingly, both
enzymes displayed a similar trend of decreasing PR protein activities
120 h postinoculation, resembling a "mycorrhiza-like" pattern.
This decrease was temporally correlated with colonization of the roots
by Trichoderma. Plant infection with mycorrhizal fungi
initiates some plant defense responses, although these do not reach
their full potential, as the latter would probably prevent
colonization. Transient increases in chitinase (38) and
peroxidase (39) activities have been detected in leek roots
during early stages of colonization by vesicular arbuscular fungi.
Vesicular arbuscular fungi are characterized by their ability to
promote growth, penetrate and colonize plant roots, and initiate marked
metabolic changes in roots (12, 39). To determine whether the observed biochemical changes correlated with structural
modifications, Trichoderma-treated root tissues were
examined at the ultrastructural level. Based on our cytological
observations, root colonization by T. harzianum involves a
sequence of events which include fungal proliferation along the
elongating root and local penetration of the epidermis. The formation
of fungal colonies at the junctions of adjacent epidermal cell walls
showing localized signs of alterations suggests that these areas are
preferential sites for replication and subsequent penetration.
Penetration of the epidermis and subsequent ingress into the outer
cortex suggest that at least small amounts of cell wall hydrolytic
enzymes, such as cellulases, are produced by the fungus to locally
weaken or loosen epidermal cell walls, thereby facilitating fungal
spread into the root tissues. However, the regular pattern of cellulose
distribution in the internal root tissues was taken as an indication
that cellulases were produced at low levels, if at all, inside the plant.
Cellular changes characterized by the deposition onto the inner cell
wall surface of callose-enriched wall appositions were typical
reactions of epidermal and cortical cells of
Trichoderma-colonized cucumber roots. This phenomenon was
even amplified by the impregnation of osmiophilic substances in the
host cell walls and in the intercellular spaces of reacting host cells.
The massive deposition of such structures at sites of attempted fungal
entry and the accumulation of osmiophilic deposits suggested that
epidermal and cortical host cells were signaled to mobilize a number of
defense strategies. Such cellular changes were apparently efficient in
preventing fungal ingress into the vascular stele, since the fungus was
seldom seen in the innermost root tissues. Fungal cells near wall
appositions frequently appeared disorganized, suggesting a fungitoxic
environment (32). Our cytochemical results provided evidence
that callose and, to a lesser extent, cellulose occurred in the wall
appositions. While callose appeared to be widely distributed over the
underlying matrix of wall appositions, cellulose was seen mainly as a
few randomly distributed molecules. In light of these observations, the
early events leading to the development of complete appositions may
involve splitting of the host cell walls followed by gradual deposition
of polysaccharides, such as callose, between the split walls.
In an attempt to determine whether enzyme-mediated wall hydrolysis was
associated with the frequent disorganization of fungal hyphae
colonizing the outer tissues in Trichoderma-infected
cucumber roots, chitin was ultrastructurally localized. An examination of the labeling pattern revealed fungal cell disorganization at a time
when chitin still occurred in the cell walls. This observation suggests
that, although chitinases are produced, they are not a primary
determinant in the expression of plant resistance to Trichoderma infection. It is more likely that the formation
of structural barriers and the synthesis of toxic substances, such as
phenolic compounds and phytoalexins, precede the production of
chitinases and other PR proteins. In agreement with this concept, the
enhanced electron density of the host cell wall and the deposition of
an osmiophilic material coating the invading hyphae in
Trichoderma-treated plants correlated well with the presence
of phenolic compounds known to stain densely upon reaction of
O-dihydroxy groups with osmium tetroxide (36).
Several studies have convincingly shown that phenolic structures can
confer high rigidity to host cell walls through peroxidase-mediated
cross-linking with preexisting wall carbohydrates, such as
hemicellulose, pectin, and callose (15).
The results presented here demonstrate that striking modifications of
epidermal and cortical cell walls, as well as deposition of newly
formed barriers, are triggered in cucumber root tissues by
colonization. These cellular changes, characterized by the deposition
of callose-enriched wall appositions onto the inner surface of the cell
walls, are apparently efficient in preventing fungal ingress into the
vascular stele. In agreement with these results, Trichoderma
treatment initiated a marked increase in peroxidase activity within
48 h after inoculation. As a general rule, peroxidase activity
increases earlier than chitinase activity in
Trichoderma-treated cucumber plants. Peroxidase may be
rapidly involved in the peroxidation of substrate molecules, leading to the accumulation of highly toxic compounds (i.e., phenolic compounds), which may contribute to resistance via their antifungal potential (41). However, these compounds may, to some extent, be toxic to the plant itself, and it seems reasonable to assume that mechanisms designed to repress peroxidase expression are activated during the
resistance process in order to maintain phenolic compounds below
phytotoxic levels. In that context, the decrease in peroxidase activity
observed at 120 h postinoculation may reflect a process elaborated
by the plant to protect itself until such activity is needed, such as
upon pathogenic attack. Like a symbiotic mycorrhizal association, the
interaction between Trichoderma and the plant may involve
molecular recognition between the two partners, resulting in the
establishment of a beneficial partnership that subsequently leads to a
decrease in the synthesis and accumulation of defense molecules. This
concept is supported by the observation that plant growth was promoted
in Trichoderma-treated plants, obviously indicating the
beneficial effect of the association.
In the present study, application of the saprophytic fungus T. harzianum to the rhizosphere of young cucumber seedlings initiated in the plants a series of morphological as well as biochemical changes
which are considered to be part of the plant defense response. To our
knowledge, this study is the first to provide evidence that T. harzianum penetrates the root system without causing extensive damage and triggers the transient elaboration of host defense reactions. As with immunization, Trichoderma-inoculated
plants may be sensitized to respond faster and to a greater extent to potential pathogen attacks. Further studies, designed to assess the
role of T. harzianum in the induction of resistance against pathogens, are in progress.
| |
ACKNOWLEDGMENT |
This research was supported by Binational Agricultural Research
and Development Fund (BARD) project IS-2880-97.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Plant Pathology and Microbiology, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel. Phone: 972-8-9481315. Fax: 972-8-9468785. E-mail: chet{at}agri.huji.ac.il.
| |
REFERENCES |
| 1.
|
Baker, R.
1989.
Improved Trichoderma spp. for promoting crop productivity.
Trends Biotech.
7:34-38.
|
| 2.
|
Benhamou, N.
1995.
Immunocytochemistry of plant defense mechanisms induced upon microbial attack.
Microsc. Res. Tech.
31:63-78[Medline].
|
| 3.
|
Benhamou, N.
1989.
Preparation and application of lectin-gold complexes, p. 95-143.
In
M. A. Hayat (ed.), Colloidal gold, principles, methods, and applications, vol. 1. Academic Press, Inc., New York, N.Y.
|
| 4.
|
Benhamou, N.
1992.
Ultrastructural detection of -1,3-glucans in tobacco root tissues infected by Phytophthora parasitica var. nicotianae using a gold-complexed tobacco -1,3-glucanase.
Physiol. Mol. Plant Pathol.
41:351-370.
|
| 5.
|
Benhamou, N.,
H. Chamberland,
G. B. Ouellette, and F. J. Pauzé.
1987.
Ultrastructural localization of -1,4-D-glucans in two pathogenic fungi and in their host tissues by means of an exoglucanase-gold complex.
Can. J. Microbiol.
33:405-417.
|
| 6.
|
Beyrle, H.
1995.
The role of phytohormones in the function and biology of mycorrhizas, p. 365-391.
In
A. Varma, and B. Hock (ed.), Mycorrhiza. Springer-Verlag KG, Berlin, Germany.
|
| 7.
|
Campbell, M. M., and B. E. Ellis.
1992.
Fungal elicitor-mediated responses in pine cell cultures: cell wall-bound phenolics.
Phytochemistry
31:737-742.
|
| 8.
|
Chet, I.
1990.
Biological control of soilborne pathogens with fungal antagonists in combination with soil treatment, p. 15-25.
In
D. Hornby, R. J. Cook, Y. Henis, W. H. Ko, A. D. Rovira, B. Schippers, and P. R. Scott (ed.), Biological control of soilborne pathogens. CAB Publishing House, New York, N.Y.
|
| 9.
|
Chet, I.
1987.
Trichoderma application, mode of action, and potential as biocontrol agent of soilborne plant pathogenic fungi, p. 137-160.
In
I. Chet (ed.), Innovative approaches to plant disease control. John Wiley & Sons, Inc., New York, N.Y.
|
| 10.
|
Dalisay, R. F., and J. A. Kuc.
1995.
Persistence of induced resistance and enhanced peroxidase and chitinase activities in cucumber plants.
Physiol. Mol. Plant Pathol.
47:315-327.
|
| 11.
|
De Meyer, G.,
J. Bigirimana,
Y. Elad, and M. Hofte.
1998.
Induced systemic resistance in Trichoderma harzianum T39 biocontrol of Botrytis cinerea.
Eur. J. Plant Pathol.
104:279-286.
|
| 12.
|
Dumas-Gaudot, E.,
J. Grenier,
V. Furlan, and A. Asselin.
1984.
Chitinase, chitosanase and -1,3 glucanase activities in Allium and Pisum roots colonized by Glomus species.
Plant Sci.
84:17-24.
|
| 13.
|
Elad, Y.,
I. Chet, and Y. Henis.
1982.
Degradation of plant pathogenic fungi by Trichoderma harzianum.
Can. J. Microbiol.
28:719-725.
|
| 14.
|
Frens, G.
1973.
Controlled nucleation for the regulation of the particle size in monodisperse gold solution.
Nat. Phys. Sci.
241:20-22.
|
| 15.
|
Fry, S. C.
1986.
Polymer-bound phenols as natural substrates of peroxidases, p. 169-182.
In
H. Greppin, C. Penel, and T. Gaspar (ed.), Molecular and physiological aspects of plant peroxidase. Université de Genève, Geneva, Switzerland.
|
| 16.
|
Ghisalberti, E. L., and K. Sivasithamparam.
1991.
Anti fungal antibiotics produced by Trichoderma spp.
Soil Biol. Biochem.
23:1011-1020.
|
| 17.
|
Haran, S.,
A. Schickler, and I. Chet.
1996.
Differential expression of Trichoderma harzianum chitinases during mycoparasitism.
Phytopathology
86:980-985.
|
| 18.
|
Haran, S.,
H. Schickler,
A. Oppenheim, and I. Chet.
1995.
New components of the chitinolytic system of Trichoderma harzianum.
Mycol. Res.
99:441-446.
|
| 19.
|
Harman, G. E., and T. Bjorkman.
1998.
Potential and existing uses of Trichoderma and Gliocladium for plant disease control and plant growth enhancement, p. 229-265.
In
C. K. Kubicek, and G. E. Harman (ed.), Trichoderma and Gliocladium. Taylor and Francis, London, England.
|
| 20.
|
Harman, G. E.,
C. K. Hayes,
M. Lorito,
R. M. Broadway,
P. A. Di,
C. Peterbauer, and A. Tronsmo.
1993.
Chitinolytic enzymes of Trichoderma harzianum: purification of chitobiosidase and endochitinase.
Phytopathology
83:313-318.
|
| 21.
|
Heath, M. C.
1996.
Plant resistance to fungi.
Can. J. Plant Pathol.
18:469-475.
|
| 22.
|
Inbar, J.,
M. Abramsky,
D. Cohen, and I. Chet.
1994.
Plant growth enhancement and disease control by Trichoderma harzianum in vegetable seedlings grown under commercial conditions.
Eur. J. Plant Pathol.
100:337-346.
|
| 23.
|
Inbar, J., and I. Chet.
1997.
Lectins and biocontrol.
Crit. Rev. Biotechnol.
17:1-20[Medline].
|
| 24.
|
Kauffmann, S.,
M. Legrand,
P. Geoffroy, and B. Fritig.
1987.
Biological function of "pathogenesis-related" proteins. Four PR proteins of tobacco have 1,3--glucanase activity.
EMBO J.
6:3209-3212[Medline].
|
| 25.
|
Kleifeld, O.
1990.
Ph.D. thesis.
Hebrew University of Jerusalem, Jerusalem, Israel.
|
| 26.
|
Kleifeld, O., and I. Chet.
1992.
Trichoderma harzianuminteraction with plants and effect on growth response.
Plant Soil
144:267-272.
|
| 27.
|
Liu, L.,
W. Kloepper, and S. Tuzun.
1995.
Induction of systemic resistance in cucumber against Fusarium wilt by plant growth promoting rhizobacteria.
Phytopathology
85:695-698.
|
| 28.
|
Meera, M. S.,
M. B. Shivanna,
K. Kageyama, and M. Hyakumachi.
1995.
Persistence of induced systemic resistance in cucumber in relation to root colonization by plant growth promoting fungal isolates.
Crop Prot.
14:123-130.
|
| 29.
|
Meera, M. S.,
M. B. Shivanna,
K. Kageyama, and M. Hyakumachi.
1994.
Plant growth promoting fungi from Zoysiagrass rhizosphere as potential inducers of systemic resistance in cucumbers.
Phytopathology
84:1399-1406.
|
| 30.
|
Meera, M. S.,
M. B. Shivanna,
K. Kageyama, and M. Hyakumachi.
1995.
Responses of cucumber to induction of systemic resistance against anthracnose by plant growth promoting fungi.
Eur. J. Plant Pathol.
101:421-430.
|
| 31.
|
Okon, Y.,
I. Chet, and Y. Henis.
1973.
Effects of lactose, ethanol and cycloheximide on the translation pattern of radioactive compounds and on Sclerotium rolfsii.
J. Gen. Microbiol.
74:251-258.
|
| 32.
|
Peng, M., and J. A. Kuc.
1992.
Peroxidase generated hydrogen peroxide as a source of antifungal activity in vitro and on tobacco leaf disks.
Phytopathology
82:696-699.
|
| 33.
|
Roberts, W. K., and C. P. Selitrennikoff.
1988.
Plant and bacterial chitinases differ in antifungal activity.
J. Gen. Microbiol.
134:169-176.
|
| 34.
|
Ruttimann, C.,
E. Schwember,
L. Salas,
D. Cullen, and R. Vicuna.
1992.
Ligninolytic enzymes of the white rot basidiomycetes Phlebia breviospora and Ceriporiopsis subvermispora.
Biotechnol. Appl. Biochem.
16:64-76.
|
| 35.
|
Samuels, G. J.
1996.
Trichoderma: a review of biology and systematics of the genus.
Mycol. Res.
100:923-935.
|
| 36.
|
Scalet, M.,
E. Crivaletto, and F. Mallardi.
1989.
Demonstration of phenolic compounds in plant tissue by an osmium-iodide post fixation procedure.
Stain Technol.
64:273-290[Medline].
|
| 37.
|
Sivan, A., and I. Chet.
1993.
Integrated control of Fusarium crown and root rot of tomato with Trichoderma harzianum in combination with methyl bromide or soil solarization.
Crop Prot.
12:380-386.
|
| 38.
|
Spanu, P.,
T. Boller,
A. Ludwig,
A. Wiemken,
A. Faccio, and P. Bonafante-Fasolo.
1989.
Chitinases in roots of mycorrhizal Allium porrum: regulation and localization.
Planta
177:447-455.
|
| 39.
|
Spanu, P., and P. Bonafante-Fasolo.
1988.
Cell wall bound peroxidase activity in roots of mycorrhizal Allium porrum.
New Phytol.
109:119-124.
|
| 40.
|
van Loon, L. C.
1985.
Pathogenesis related proteins.
Plant Mol. Biol.
4:111-116.
|
| 41.
|
Ward, E. W. B.
1986.
Biochemical mechanisms involved in resistance of plants to fungi, p. 107-131.
In
J. A. Baily (ed.), Biology and molecular biology of plant-pathogen interactions. Springer-Verlag KG, Berlin, Germany.
|
| 42.
|
Wei, G.,
J. W. Kloepper, and S. Tuzun.
1992.
Induction of systemic resistance of cucumber to Colletotrichum orbiculare by select strains of plant growth promoting rhizobacteria.
Phytopathology
81:1508-1512.
|
Applied and Environmental Microbiology, March 1999, p. 1061-1070, Vol. 65, No. 3
0099-2240/99
Top
Abstract
Introduction
