APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1999, p.

929935
0099-2240/99/$04.0010
Copyright 1999, American Society for Microbiology. All Rights Reserved.

Vol. 65, No. 3

Role of the Trichoderma harzianum Endochitinase Gene,

ech42, in Mycoparasitism
S,1
CAROLINA CARSOLIO,1 NICOLE BENHAMOU,2 SHOSHAN HARAN,3 CARLOS CORTE
RREZ,1 ILAN CHET,3 AND ALFREDO HERRERA-ESTRELLA1*
ANA GUTIE
Centro de Investigacio
n y Estudios Avanzados, Plant Biotechnology and Genetic Engineering Unit,
Irapuato, Mexico1; Universite Laval, Ste. Foy, Quebec, Canada2; and Otto Warburg
Center for Agricultural Biotechnology, Faculty of Agriculture, The Hebrew
University of Jerusalem, Rehovot, Israel3
Received 21 May 1998/Accepted 3 December 1998

The role of the Trichoderma harzianum endochitinase (Ech42) in mycoparasitism was studied by genetically
manipulating the gene that encodes Ech42, ech42. We constructed several transgenic T. harzianum strains carrying multiple copies of ech42 and the corresponding gene disruptants. The level of extracellular endochitinase activity when T. harzianum was grown under inducing conditions increased up to 42-fold in multicopy strains as
compared with the wild type, whereas gene disruptants exhibited practically no activity. The densities of chitin
labeling of Rhizoctonia solani cell walls, after interactions with gene disruptants were not statistically significantly different than the density of chitin labeling after interactions with the wild type. Finally, no major differences
in the efficacies of the strains generated as biocontrol agents against R. solani or Sclerotium rolfsii were observed
in greenhouse experiments.
Trichoderma harzianum is a saprophytic fungus which is used
as a biological control agent against a wide range of economically important aerial and soilborne plant pathogens (6, 25).
The mycoparasitic activity of Trichoderma spp. may be due to
antibiosis (13), competition (6), production of cell wall-degrading enzymes (6, 29), or a combination of these antagonistic
activities.
Mycoparasitism is a complex process in which a Trichoderma
species grows chemotropically toward its host and attaches to
and coils around the host hyphae, sometimes penetrating them
(6). Partial degradation of the host cell wall is normally observed in later stages of the parasitic process. The effects of cell
wall-degrading enzymes on the host have been observed by using different ultrastructural and/or histochemical approaches.
Staining with fluorescein isothiocyanate-conjugated lectins or
calcofluor has indicated that localized cell wall lysis occurs at
points of contact between the antagonist and its host (9). These
data, together with recent electron microscope observations,
have led to the hypothesis that during the interaction of Trichoderma spp. with either Sclerotium rolfsii or Rhizoctonia solani,
the host cell walls are enzymatically digested by the parasite
(2, 5, 8, 9). The support for this hypothesis includes the finding that Trichoderma spp. produce extracellular b-(1,3)-glucanases, chitinases, lipases, and proteases when they are grown
on cell walls of pathogenic fungi. In addition, several lines of
evidence have shown that the production of some lytic enzymes
is induced during the parasitic interaction between Trichoderma spp. and some pathogenic fungi (for a review see reference 14).
The chitinolytic system of T. harzianum consists of five to
seven distinct enzymes, depending on the strain (15). In the
best-characterized Trichoderma isolate (isolate TM), the system is apparently composed of two b-(1,4)-N-acetylglucosaminidases (102 and 73 kDa) and four endochitinases (52,

42, 33, and 31 kDa) (15). Different components of the chitinolytic system of T. harzianum probably involve complementary modes of action of the component enzymes. However, the
entire system might be required for maximum efficacy (21).
The most interesting individual enzyme of the complex is the
42-kDa endochitinase (Ech42), which can hydrolyze in vitro
Botrytis cinerea cell walls and inhibits spore germination and
germ tube elongation of various fungi (7, 2123, 29). The corresponding gene (ech42) is strongly induced during fungusfungus interactions and when the fungus is grown in the presence of autoclaved mycelia of several fungi or with colloidal
chitin as the sole carbon source (3, 12). ech42 expression is
repressed by glucose and is increased by exposure to light and
by nutritional stress conditions (3, 12).
Our working hypothesis is that endochitinase Ech42 plays a
key role in cell wall degradation and thus in biocontrol. We
tested this hypothesis by making some strains that overproduce
this enzyme and other strains that lack it completely and then
testing these strains for efficacy as biocontrol agents.
MATERIALS AND METHODS
Strains and plasmids. T. harzianum IMI206040 was used in this work. Escherichia coli JM103 (27) and MC1061 (4) were used for DNA manipulation. The
plasmids used were pBluescript (Stratagene), pSPORT (Gibco-BRL, Madison,
Wis.), and pHATa, which carries the E. coli hygromycin phosphotransferase
(hph) gene as a dominant selectable marker (16).
Plasmid construction. A 9-kb BamHI fragment from the ech42 genomic clone
in lEMBL3 (3) was subcloned into pBluescript, generating plasmid pQuiA9. The
inserted fragment contained the entire coding region for Ech42 plus approximately 2 kb of the 39 region and 5 kb of the 59 region. Plasmid pQuiA9 was used
to transform T. harzianum protoplasts as previously described (16, 20).
A 1.4-kb expression cassette from plasmid pCB1004 (Fungal Genetics Stock
Center) carrying the E. coli hph gene under the control of the trpC promoter
from Aspergillus nidulans was digested with HpaI and ligated into the MscI site of
pQuiA2.3 (3), generating plasmid pQuiA2.3D. In this way 228 bp from the ech42
gene was replaced by the hygromycin resistance cassette.
PCR amplification. PCR were carried out with T. harzianum genomic DNA
from the different transformants obtained with plasmid pQuiA2.3D. Primers
were designed so that homologous recombination or ectopic integration resulted
in different band patterns. Briefly, the forward primer (59 GGACCAGGTGCT
GTT 39) hybridized on the 59 side of the gene (ech42) but outside the fragment
contained in pQuiA2.3D, and the reverse primer (59 TAGTTGAGACCGCT
TCG 39) hybridized in the coding region downstream of the insertion site of the

* Corresponding author. Mailing address: Centro de Investigacio

n y
Estudios Avanzados, Unidad Irapuato, A.P. 629, 36500 Irapuato, Gto.,
Mexico. Phone: 52 462 39658. Fax: 52 462 45849. E-mail: aherrera
@irapuato.ira.cinvestav.mx.
929

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CARSOLIO ET AL.

cassette. Taq polymerase (Gibco-BRL) was used for all amplifications. The following PCR program was used: a hot start consisting of 5 min at 95C; 25 cycles
consisting of 1 min at 94C, 2 min at 60C, and 3 min at 72C; and a final extension period consisting of 7 min at 72C. A 4.2-kb band was expected for
disruptants, whereas transformants in which integration had occurred ectopically
should have had a 3.0-kb fragment.
DNA and RNA manipulations. Fungal chromosomal DNA was isolated as
previously described (26). All other DNA manipulations were performed by
using standard techniques (27). Fungal RNA was isolated essentially by the
protocol described by Jones et al. (17). Southern blotting and Northern blotting
were performed by using standard procedures (27).
Growth conditions. For ech42 induction, T. harzianum strains were grown as
previously described (33) except that after the second centrifugation, mycelia
were transferred to minimal medium (MM) containing 0.75% colloidal chitin as
the sole carbon source. Culture media were recovered by filtration after 48 h of
growth, frozen in liquid nitrogen, and kept at 270C until they were used.
To establish growth curves, strains were grown as previously described (33),
but after the second centrifugation mycelia were transferred to fresh MM supplemented with 2% glycerol or 2% glycerol plus 0.75% colloidal chitin. Mycelia
were collected at different times, freeze-dried, and weighed.
Chitinase assays. Chitinase activities were determined by using culture filtrates. Culture filtrates were freeze-dried and resuspended in 1.5 ml of chitinase
reaction buffer (50 mM sodium acetate, 0.15 mg of phenylmethylsulfonyl fluoride
per ml, 10 mM EDTA; pH 5.0), and activity was determined as previously described (3).
Chitinase activity as determined by SDS-PAGE. Protein samples (20 ml) from
T. harzianum were freeze-dried, resuspended in 3 ml of deionized water, dialyzed
against H2O for 36 h at 4C, frozen, and lyophilized again. The samples were
resuspended in 300 to 500 ml of H2O, divided into aliquots, frozen, and kept at
270C until they were used. One to ten micrograms of each sample was resuspended in Laemmli buffer (19) without b-mercaptoethanol and subjected to
sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) without boiling in 0.7-mm-thick 4% acrylamide stacking gels and 10% acrylamide
separating gels. After electrophoresis the SDS was removed by using the caseinEDTA procedure (24) as modified by Haran et al. (15). The gels were overlaid
with 2 ml of 1% low-melting-point agarose in acetate buffer containing 300 mg of
4-methylumbelliferyl b-D-N,N9,N0-triacetylchitotriose and incubated at 37C for
10 min, and the bands visualized under short-wavelength UV light (32).
Western blot analysis. Proteins were electrophoresed in polyacrylamide-SDS
gels as described above for the enzyme activity analysis, except that the samples
were boiled in the presence of b-mercaptoethanol. The gels were electrotransferred to Immobilon membranes. The blots were sequentially treated with rabbit
anti-Ech42 antibodies and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G. Phosphatase color was developed by incubating the preparations with BCIP (5-bromo-4-chloro-3-indolyl phosphate) and nitroblue tetrazolium.
TEM. For electron microscope investigations, mycelial disks (diameter, 5 mm)
that were cut from actively growing colonies of both fungi were placed 3 cm apart
on the surfaces of petri dishes containing freshly prepared potato dextrose agar
(PDA). The petri dishes were incubated at 25C with continuous light. Mycelial
samples from the interaction regions were collected 4 and 16 h after contact, as
estimated visually. Samples were fixed with 3% (vol/vol) glutaraldehyde in 0.1 M
sodium cacodylate buffer (pH 7.2) overnight at 4C and postfixed with 1%
(wt/vol) osmium tetroxide in the same buffer for 1 h at 4C. The samples were
dehydrated in a graded ethanol series and embedded in Epon 812. Ultrathin
sections (thickness, 70 nm) were cut with a diamond knife, were collected on
Formvar-coated nickel grids, and were either contrasted with uranyl acetate and
lead citrate for direct examination with a JEOL model 1200 EX transmission
electron microscope (TEM) at 80 kV or processed for cytochemical labeling.
Three samples per sampling time were examined by using an average of 10 grid
squares per sample.
Cytochemical labeling. Colloidal gold (average particle diameter, 12 nm) was
prepared as described by Frens (11). To study the distribution of chitin, a linear
polysaccharide consisting of b-1,4-linked N-acetylglucosamine residues, wheat
germ agglutinin (WGA), a lectin with N-acetylglucosamine-binding specificity
(1), was used in a two-step procedure. Ovomucoid, a high-molecular-weight
glycoprotein, was used as a second-step reagent due to its specific binding affinity
for WGA. The glycoprotein was complexed with colloidal gold at pH 5.4. Sections were incubated with 1 drop of phosphate-buffered saline solution (PBS)
(pH 7.2) for 5 min and then with 1 drop of WGA (12.5 mg/ml in PBS) for 30 to
60 min at room temperature in a moist chamber. After thorough washing with
PBS, the sections were incubated with the ovomucoid-gold complex (1:60 in
PBS-polyethylene glycol 20000). The sections were washed with PBS, rinsed with
distilled water, and contrasted with uranyl acetate and lead citrate. The specificity of the labeling was assessed by (i) incubation with WGA to which N-N9N0-triacetylchitotriose (1 mg/ml in PBS) had been added previously; (ii) incubation with WGA, then with uncomplexed ovomucoid, and finally with the
ovomucoid-gold complex; and (iii) direct incubation with the gold-complexed
ovomucoid with the lectin step omitted.
Quantification of labeling. The density of labeling obtained with the WGAovomucoid-gold complex was determined by counting the number of gold particles per square micrometer. Areas were determined by the point counting

APPL. ENVIRON. MICROBIOL.

FIG. 1. DNA analysis of the gene disruption candidates: PCR-amplified

products. Samples (100 ng) of total DNA from different transformants were
subjected to PCR and agarose gel electrophoresis. Lane 1, DQ-3; lane 2, DQ-1;
lane 3, DQ-2; lane 4, another DQ transformant not carrying an ech42 disruption;
lane 5, wild type; lane M, molecular size standards. The expected sizes of the
bands corresponding to wild type ech42 (WT) and disrupted ech42 (DQ) are
indicated on the right.
method of Weibel (34) by using negatives of electron micrographs projected onto
a lattice. The amount of labeling in a specified wall area (Sa) was estimated by
counting the number of gold particles (Ni) on a photographic enlargement. The
density of labeling (Ns) was calculated as follows: Ns 5 Ni/Sa, where Ns is the
number of gold particles per unit surface.
Greenhouse experiments. Experiments were carried out in a sandy loam soil
consisting of 82% sand, 2% silt, 15% clay, and 0.4% organic matter (pH 7.4) and
having a moisture-holding capacity of 12%. The temperature ranged from 27 to
30C. Daily irrigation was provided. Trichoderma sp. was added to the soil as a
wheat bran-peat mixture (0.5%, wt/wt). Chopped potato soil containing R. solani
was prepared as described by Ko and Hora (18) and was used for soil infestation.
Soil was artificially infested with S. rolfsii by adding sclerotia from a 10-day-old
dried synthetic medium agar culture. Cotton (Gossypium barbardenso L.) seedlings were used in the experiments, which were performed in six replicates by
using plastic pots, each containing 0.5 kg of soil (10 plants/replicate). The complete experiment was repeated twice.

RESULTS
Overexpression of ech42. Ech42 is thought to be a very important component of the T. harzianum chitinolytic system in
terms of mycoparasitism (3, 7, 2123, 29). We transformed
T. harzianum with plasmid pQuiA9 in order to generate strains
carrying multiple copies of ech42. Plasmid pHATa was cotransformed as a marker, and protoplasts were selected on the
basis of hygromycin resistance. Transformants were subjected
to two rounds of monosporic selection. A Southern analysis of
12 independent transformants revealed that there was ectopic
integration of the construct, which left the original gene intact
(data not shown). Six transformants carrying one to six extra
copies of ech42 were selected and were designated the QL
transformants.
ech42 disruption. We transformed T. harzianum with plasmid pQuiA2.3D, a derivative of pQuiA2.3 (3) in which a small
portion (228 bp) of the ech42 coding region was replaced by a
cassette conferring hygromycin resistance. Transformants were
selected on the basis of hygromycin resistance, and 3 of 70
transformants yielded the product expected if homologous insertion of the disrupting construct had occurred (Fig. 1, lanes
1 through 3). Two of these transformants (DQ-1 and DQ-2)
(Fig. 1, lanes 2 and 3) appeared to be homogeneous, whereas
the third (DQ-3) (lane 1) apparently was chimeric and carried
both disrupted and wild type nuclei. These three cultures were
subjected to a third round of monosporic culturing, and gene
disruption was confirmed by Southern analysis following digestion of genomic DNA with SalI. A 2.3-kb band was expected
from the wild type and a 3.5-kb band was expected from the
disruptant when the cultures were hybridized with an internal
fragment of the ech42 coding region as the probe. All three
putative disruptants had the expected 3.5-kb band (Fig. 2, lanes

ROLE OF ech42 IN TRICHODERMA MYCOPARASITISM

VOL. 65, 1999

FIG. 2. Southern analysis. Total DNA was extracted, digested with SalI, and
probed with a HindIII fragment from the ech42 coding region. The expected sizes
of the bands corresponding to wild type ech42 (WT) and disrupted ech42 (DQ)
are indicated on the left, and the positions of molecular size standards are
indicated on the right.

1 through 3) and were clearly different from the wild type (Fig.
2, lane 4).
Chitinase expression in the transformed Trichoderma. We
tested five multicopy transformants and the three disruptants
to determine the levels of protein and chitinase activity. Strains
were grown by using chitin as the sole carbon source, and
samples were collected at the time when the highest level of
activity was detected in the wild-type strain. The QL transformants had chitinase activities that were up to 42 times higher
than the activity of the wild type (Table 1). The disruptants
exhibited virtually no activity.
Protein samples from the disruptants and three of the QL
transformants were tested for chitinase activity by SDS-PAGE
(Fig. 3). No activity was observed with DQ-1, DQ-2, or DQ-3,
while all of the QL transformants exhibited much greater activity than wild-type T. harzianum exhibited. For the QL transformants two bands appeared close together when a single
band was expected.
Protein samples were subjected to a Western blot analysis by
using a rabbit polyclonal antibody, and a single band at the
expected molecular weight was detected (Fig. 4); no protein
was detected in the two disruptants analyzed (Fig. 4, lanes 5
and 6). Densitometric analysis of the Western blot indicated

931

FIG. 3. Chitinase expression in transformed Trichoderma sp.: detection of

extracellular chitinolytic activity from T. harzianum transformed strains. Protein
samples were subjected to SDS-PAGE and renatured, and the activity was
revealed by using 4-methylumbelliferyl b-D-N,N9,N0-triacetylchitotriose as the
substrate. Lane 1, DQ-1; lane 2, DQ-2; lane 3, DQ-3; lane 4, wild type; lane 5,
QL-9; lane 6, QL-7; lane 7, QL-10. Schematic representations of the different
constructs used to transform T. harzianum are shown at the top.

that the QL transformants (Fig. 4, lanes 1 through 3) secreted

six times as much chitinase as the wild type secreted (lane 4).
Quantification of chitin labeling of R. solani cell walls. Direct confrontation experiments performed with some of the
transgenic Trichoderma strains and R. solani were carried out,
and the results were studied at the microscopic level. The
damage to the R. solani cell walls caused by QL-9, DQ-1, and
the wild type was evaluated microscopically by labeling chitin
with the WGA-ovomucoid-colloidal gold complex (Fig. 5). As
soon as 4 h after the initial contact, slight degradation of the
Rhizoctonia cell wall by the wild type was observed (Fig. 5A).
The ability of disruptant DQ-1 to attack the cell wall-bound
chitin did not seem to be affected (Fig. 5B). In contrast, markedly greater cell wall alterations were observed with overproducing clone QL-9 at the same time (Fig. 5C, arrowheads).
These effects were distinct when the density of labeling was

TABLE 1. Chitinase activities secreted by transformed

and wild-type T. harzianum strains
Strain

Chitinase activity
(pmol of MU/min/mg
of protein)a

IMI206040 .............................................................................
27
QL-5....................................................................................... 620
QL-7....................................................................................... 1,200
QL-9....................................................................................... 910
QL-10..................................................................................... 570
QL-11..................................................................................... 850
DQ-1.......................................................................................
2.1
DQ-2.......................................................................................
3.4
DQ-3.......................................................................................
5.1
a
The results shown are the results of a representative experiment. Similar
results were obtained in three independent experiments. MU, 4-methylumbelliferone.

FIG. 4. Western blot of extracellular proteins from T. harzianum transformed strains. A 2.5-mg sample of protein from each strain (lanes 1 through 6)
or 20 ng of purified recombinant Ech42 from E. coli (lane 7) was subjected to
SDS-PAGE and transferred onto a nylon membrane. An antibody against Ech42
was used for immunodetection. Lane 1, QL-10; lane 2, QL-9; lane 3, QL-7; lane
4, wild type; lane 5, DQ-2; lane 6, DQ-1; lane 7, recombinant Ech42 from E. coli.
Schematic representations of the different constructs used to transform T. harzianum are shown at the top.

932

CARSOLIO ET AL.

APPL. ENVIRON. MICROBIOL.

FIG. 5. TEM micrographs of cultures containing either wild-type or transformed T. harzianum and R. solani. Chitin was labeled with the WGA-ovomucoid-gold
complex. (A) Wild type, 4 h. (B) DQ-1, 4 h. (C) QL-9, 4 h. (D) QL-9, 16 h. T, Trichoderma cell; R, Rhizoctonia cell; CW, Rhizoctonia cell wall. The arrowheads indicate
points where R. solani cell wall degradation is apparent.

VOL. 65, 1999

ROLE OF ech42 IN TRICHODERMA MYCOPARASITISM

933

TABLE 2. Densities of labeling obtained with the

WGA-ovomucoid-gold complex for cell walls of
R. solani during interactions with wild-type
and transformed T. harzianum strains
Strain

Wild type
QL-9
DQ-1

FIG. 6. Plant disease control by wild-type and transformed T. harzianum in

greenhouse experiments. Columns for each series (R. solani, S. rolfsii) marked
with different letters are significantly different (P 5 0.05), as determined by
Duncans multiple range test. Control, R. solani or S. rolfsii in soil; QL-9, pathogen plus QL-9; DQ-1, pathogen plus DQ-1; WT, pathogen plus wild-type T. harzianum.

used as an indirect measurement of the chitin content of the

cell wall (Table 2). By 16 h the plasma membrane appeared to
be altered and the cell wall was clearly degraded by all strains
(Fig. 5D). Although at 4 h there was a tendency toward a lower
level of chitin labeling in the Rhizoctonia cell walls when the
Rhizoctonia cells were confronted with QL-9, this tendency was
statistically significant only at 16 h. The particle counts in the
DQ-1 micrographs were similar to the particle counts in the
wild-type micrographs (Table 2). We measured ech42 gene
expression in wild-type and QL-9 strains grown on PDA alone
and in the presence of the pathogen. The basal level of gene
expression was much higher in QL-9 than in the wild type,
although no obvious induction was observed (data not shown).
Greenhouse experiments. In the greenhouse tests (Fig. 6)
the disease incidence when wild-type T. harzianum was used to
control R. solani was 33%, and the disease incidence when
wild-type T. harzianum was used to control S. rolfsii was 25%;
the values for DQ-1 were similar. QL-9 significantly reduced
disease incidence due to R. solani (14%) but not disease incidence due to S. rolfsii. Similar results were obtained with other
QL transformants (data not shown).
Growth curves. We grew wild-type T. harzianum in minimal
medium containing glycerol supplemented or not supplemented with chitin as an inducer of ech42 expression. Under these
conditions, ech42 expression was detected by 72 h and reached
a maximum level at 110 h; no ech42 expression was observed
when T. harzianum was grown in the absence of chitin (data
not shown). Two QL transformants, the DQ-1 disruptant, and
the wild-type strain were cultured as described above, and their
growth curves were determined (Fig. 7). After 85 h in the
chitin-containing medium, the QL transformants had gained

Density of gold particles

(no. of particles per mm2) aftera:
4 hb

16 h

140 6 16c
98 6 12
120 6 7

81 6 12
43 6 18
65 6 12

a
Densities were determined by counting the number of gold particles in 5 mm2
of cell wall area for 20 micrographs for each strain.
b
Time after the initial contact.
c
Mean 6 standard deviation.

more weight than DQ-1 or the wild type, but the growth of the
QL transformants declined faster later on. In every case the
loss of biomass was accelerated in the presence of chitin. This
effect was even more pronounced with the two QL transformants. In the case of the disruptant, the decline in growth was
slower. We also grew Trichoderma sp. on PDA plates and MM
plates amended with either glucose or chitin. In all cases, the
radial growth of the transformants was not significantly different from the radial growth of the wild type (data not shown).
DISCUSSION
Recently, it has been proposed that the complex chitinolytic
system of T. harzianum is a key component in mycoparasitism
(3, 7). In an attempt to determine the role of Ech42 in the
mycoparasitic process, we constructed transgenic Trichoderma
strains carrying multiple copies of ech42 and deletion mutants.
Expression of ech42 was directed by its own promoter, and we
expected that in most transformants, the gene would be regulated both in time and space as it is in the wild type, resulting
in production of the enzyme where and when it is normally
required, albeit in larger quantities.
There was almost no chitinase activity in the gene disruptants, while the QL transformants exhibited much higher levels
of chitinase activity than the wild type exhibited (Table 1). The
activity observed corresponded to two protein bands on SDSPAGE gels, although both bands may have originated from a
single protein because the two activities increased in parallel in
the overexpressants and neither activity was detected in the
disruptants. These bands may correspond to two different conformations of the same protein. When the protein samples
were boiled in the presence of b-mercaptoethanol for the
Western blot analysis, a single protein band that reacted with
the anti-Ech42 antibodies was detected, supporting this hypothesis. Our observations differ from observations made in

FIG. 7. Growth curves for transformed T. harzianum strains. Strains were grown in MM containing glycerol (F) or MM containing glycerol and chitin (E).

934

CARSOLIO ET AL.

previous studies (15, 28), in which activity bands with different

molecular weights were detected in other T. harzianum strains.
In other studies we have observed that the strain used in our
work secretes at least two exochitinases (unpublished observations). However, the conditions used in our assays favored
Ech42 activity.
Direct confrontation experiments performed with the Trichoderma QL and DQ transformants and R. solani did not reveal
obvious differences in the capacities of these organisms to overgrow Rhizoctonia sp. compared to the nontransformed strain in
vitro, except for slightly wider areas of apparent lysis in the
contact lines when the multicopy transformants were used
(data not shown). Electron microscope investigations of the
damage to Rhizoctonia cell walls caused by DQ-1 and the wild
type revealed no significant differences in the chitin content.
However, there were significant differences between QL-9 and
the wild type. These differences were not proportional to the
differences in chitinase activity and supported the hypothesis
that the combined action of a set of chitinases is required for
efficient degradation of chitin in the fungal cell wall. These results also suggest that in the complex chitinolytic system specific enzymatic activities are redundant. In all cases major alterations, including retraction of the plasma membrane and
aggregation of the cytoplasm, occurred prior to cell wall degradation, suggesting that an alternative mechanism may be the
primary determinant in the mycoparasitic process of the Trichoderma strains analyzed.
We were surprised that there was no significant difference
between the disruptants and the wild type in the greenhouse
biocontrol tests (Fig. 6). An analogous situation has been described for the plant-pathogenic fungus Cochliobolus carbonum, in which disruption of an endopolygalacturonase gene
did not affect pathogenicity (30). One possible explanation is
that other enzymes of the chitinolytic system of Trichoderma
sp. are sufficient for control of both R. solani and S. rolfsii. In
addition, it has been shown that the lack of a certain protein
can be compensated for by altering the levels of other proteins
with similar activities (31).
Treatment with the transgenic Trichoderma strain that produced the highest chitinase levels in vitro reduced the disease
incidence 9 to 19% compared to treatment with the wild type
(Fig. 6). In contrast, we previously observed a fivefold reduction in the incidence of disease caused by R. solani when we
used a transgenic Trichoderma strain carrying multiple copies
of the mycoparasitism-related gene prb1 (10). We suggest that
the levels of chitinases naturally secreted by Trichoderma species are high enough for efficient biocontrol of the phytopathogenic fungi tested. Thus, an increase in the production of one
of the chitinases would not dramatically affect the mycoparasitic capacity.
Taken together, our data demonstrate that expression of
ech42 is one part of a series of responses of Trichoderma
species to the presence of a potential host and that this gene is
not essential for effective plant disease control.
ACKNOWLEDGMENTS
We thank Jose Ruz-Herrera, Luis Herrera-Estrella, and Benjamin
Horwitz for critically reading the manuscript. We thank G. E. Harman
for donating the anti-Ech42 antibodies.
This work was supported in part by grant 3527P-N9607 from
CONACYT and by grant C/2446-1 from IFS to A.H.-E, by a grant
from the German-Israel Foundation to I.C., and by CONACYT doctoral fellowships to C. Carsolio and C. Cortes.
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