APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/99/$04.0010

Mar. 1999, p. 929–935 Vol. 65, No. 3

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Role of the Trichoderma harzianum Endochitinase Gene,ech42, in Mycoparasitism

CAROLINA CARSOLIO,1 NICOLE BENHAMOU,2 SHOSHAN HARAN,3 CARLOS CORTES,1

ANA GUTIERREZ,1 ILAN CHET,3 AND ALFREDO HERRERA-ESTRELLA1*

Centro de Investigacion 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 HebrewUniversity 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 geneticallymanipulating the gene that encodes Ech42, ech42. We constructed several transgenic T. harzianum strains carry-ing multiple copies of ech42 and the corresponding gene disruptants. The level of extracellular endochitinase ac-tivity when T. harzianum was grown under inducing conditions increased up to 42-fold in multicopy strains ascompared with the wild type, whereas gene disruptants exhibited practically no activity. The densities of chitinlabeling of Rhizoctonia solani cell walls, after interactions with gene disruptants were not statistically signifi-cantly different than the density of chitin labeling after interactions with the wild type. Finally, no major differencesin the efficacies of the strains generated as biocontrol agents against R. solani or Sclerotium rolfsii were observedin greenhouse experiments.

Trichoderma harzianum is a saprophytic fungus which is usedas a biological control agent against a wide range of econom-ically important aerial and soilborne plant pathogens (6, 25).The mycoparasitic activity of Trichoderma spp. may be due toantibiosis (13), competition (6), production of cell wall-degrad-ing enzymes (6, 29), or a combination of these antagonisticactivities.

Mycoparasitism is a complex process in which a Trichodermaspecies grows chemotropically toward its host and attaches toand coils around the host hyphae, sometimes penetrating them(6). Partial degradation of the host cell wall is normally ob-served in later stages of the parasitic process. The effects of cellwall-degrading enzymes on the host have been observed by us-ing different ultrastructural and/or histochemical approaches.Staining with fluorescein isothiocyanate-conjugated lectins orcalcofluor has indicated that localized cell wall lysis occurs atpoints of contact between the antagonist and its host (9). Thesedata, together with recent electron microscope observations,have led to the hypothesis that during the interaction of Tricho-derma 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 find-ing that Trichoderma spp. produce extracellular b-(1,3)-glu-canases, chitinases, lipases, and proteases when they are grownon cell walls of pathogenic fungi. In addition, several lines ofevidence have shown that the production of some lytic enzymesis induced during the parasitic interaction between Tricho-derma spp. and some pathogenic fungi (for a review see refer-ence 14).

The chitinolytic system of T. harzianum consists of five toseven distinct enzymes, depending on the strain (15). In thebest-characterized Trichoderma isolate (isolate TM), the sys-tem is apparently composed of two b-(1,4)-N-acetylglucos-aminidases (102 and 73 kDa) and four endochitinases (52,

42, 33, and 31 kDa) (15). Different components of the chiti-nolytic system of T. harzianum probably involve complemen-tary modes of action of the component enzymes. However, theentire system might be required for maximum efficacy (21).The most interesting individual enzyme of the complex is the42-kDa endochitinase (Ech42), which can hydrolyze in vitroBotrytis cinerea cell walls and inhibits spore germination andgerm tube elongation of various fungi (7, 21–23, 29). The cor-responding gene (ech42) is strongly induced during fungus-fungus interactions and when the fungus is grown in the pres-ence of autoclaved mycelia of several fungi or with colloidalchitin as the sole carbon source (3, 12). ech42 expression isrepressed by glucose and is increased by exposure to light andby nutritional stress conditions (3, 12).

Our working hypothesis is that endochitinase Ech42 plays akey role in cell wall degradation and thus in biocontrol. Wetested this hypothesis by making some strains that overproducethis enzyme and other strains that lack it completely and thentesting these strains for efficacy as biocontrol agents.

MATERIALS AND METHODS

Strains and plasmids. T. harzianum IMI206040 was used in this work. Esch-erichia coli JM103 (27) and MC1061 (4) were used for DNA manipulation. Theplasmids 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 clonein lEMBL3 (3) was subcloned into pBluescript, generating plasmid pQuiA9. Theinserted fragment contained the entire coding region for Ech42 plus approxi-mately 2 kb of the 39 region and 5 kb of the 59 region. Plasmid pQuiA9 was usedto transform T. harzianum protoplasts as previously described (16, 20).

A 1.4-kb expression cassette from plasmid pCB1004 (Fungal Genetics StockCenter) carrying the E. coli hph gene under the control of the trpC promoterfrom Aspergillus nidulans was digested with HpaI and ligated into the MscI site ofpQuiA2.3 (3), generating plasmid pQuiA2.3D. In this way 228 bp from the ech42gene was replaced by the hygromycin resistance cassette.

PCR amplification. PCR were carried out with T. harzianum genomic DNAfrom the different transformants obtained with plasmid pQuiA2.3D. Primerswere designed so that homologous recombination or ectopic integration resultedin different band patterns. Briefly, the forward primer (59 GGACCAGGTGCTGTT 39) hybridized on the 59 side of the gene (ech42) but outside the fragmentcontained in pQuiA2.3D, and the reverse primer (59 TAGTTGAGACCGCTTCG 39) hybridized in the coding region downstream of the insertion site of the

* Corresponding author. Mailing address: Centro de Investigacion yEstudios 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.

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cassette. Taq polymerase (Gibco-BRL) was used for all amplifications. The fol-lowing PCR program was used: a hot start consisting of 5 min at 95°C; 25 cyclesconsisting of 1 min at 94°C, 2 min at 60°C, and 3 min at 72°C; and a final ex-tension period consisting of 7 min at 72°C. A 4.2-kb band was expected fordisruptants, whereas transformants in which integration had occurred ectopicallyshould have had a 3.0-kb fragment.

DNA and RNA manipulations. Fungal chromosomal DNA was isolated aspreviously described (26). All other DNA manipulations were performed byusing standard techniques (27). Fungal RNA was isolated essentially by theprotocol described by Jones et al. (17). Southern blotting and Northern blottingwere performed by using standard procedures (27).

Growth conditions. For ech42 induction, T. harzianum strains were grown aspreviously described (33) except that after the second centrifugation, myceliawere transferred to minimal medium (MM) containing 0.75% colloidal chitin asthe sole carbon source. Culture media were recovered by filtration after 48 h ofgrowth, frozen in liquid nitrogen, and kept at 270°C 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 sup-plemented with 2% glycerol or 2% glycerol plus 0.75% colloidal chitin. Myceliawere collected at different times, freeze-dried, and weighed.

Chitinase assays. Chitinase activities were determined by using culture fil-trates. Culture filtrates were freeze-dried and resuspended in 1.5 ml of chitinasereaction buffer (50 mM sodium acetate, 0.15 mg of phenylmethylsulfonyl fluorideper ml, 10 mM EDTA; pH 5.0), and activity was determined as previously de-scribed (3).

Chitinase activity as determined by SDS-PAGE. Protein samples (20 ml) fromT. harzianum were freeze-dried, resuspended in 3 ml of deionized water, dialyzedagainst H2O for 36 h at 4°C, frozen, and lyophilized again. The samples wereresuspended in 300 to 500 ml of H2O, divided into aliquots, frozen, and kept at270°C until they were used. One to ten micrograms of each sample was resus-pended in Laemmli buffer (19) without b-mercaptoethanol and subjected tosodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) with-out boiling in 0.7-mm-thick 4% acrylamide stacking gels and 10% acrylamideseparating gels. After electrophoresis the SDS was removed by using the casein-EDTA procedure (24) as modified by Haran et al. (15). The gels were overlaidwith 2 ml of 1% low-melting-point agarose in acetate buffer containing 300 mg of4-methylumbelliferyl b-D-N,N9,N0-triacetylchitotriose and incubated at 37°C for10 min, and the bands visualized under short-wavelength UV light (32).

Western blot analysis. Proteins were electrophoresed in polyacrylamide-SDSgels as described above for the enzyme activity analysis, except that the sampleswere boiled in the presence of b-mercaptoethanol. The gels were electrotrans-ferred to Immobilon membranes. The blots were sequentially treated with rabbitanti-Ech42 antibodies and alkaline phosphatase-conjugated goat anti-rabbit im-munoglobulin G. Phosphatase color was developed by incubating the prepara-tions with BCIP (5-bromo-4-chloro-3-indolyl phosphate) and nitroblue tetrazo-lium.

TEM. For electron microscope investigations, mycelial disks (diameter, 5 mm)that were cut from actively growing colonies of both fungi were placed 3 cm aparton the surfaces of petri dishes containing freshly prepared potato dextrose agar(PDA). The petri dishes were incubated at 25°C with continuous light. Mycelialsamples from the interaction regions were collected 4 and 16 h after contact, asestimated visually. Samples were fixed with 3% (vol/vol) glutaraldehyde in 0.1 Msodium cacodylate buffer (pH 7.2) overnight at 4°C and postfixed with 1%(wt/vol) osmium tetroxide in the same buffer for 1 h at 4°C. The samples weredehydrated in a graded ethanol series and embedded in Epon 812. Ultrathinsections (thickness, 70 nm) were cut with a diamond knife, were collected onFormvar-coated nickel grids, and were either contrasted with uranyl acetate andlead citrate for direct examination with a JEOL model 1200 EX transmissionelectron microscope (TEM) at 80 kV or processed for cytochemical labeling.Three samples per sampling time were examined by using an average of 10 gridsquares per sample.

Cytochemical labeling. Colloidal gold (average particle diameter, 12 nm) wasprepared as described by Frens (11). To study the distribution of chitin, a linearpolysaccharide consisting of b-1,4-linked N-acetylglucosamine residues, wheatgerm agglutinin (WGA), a lectin with N-acetylglucosamine-binding specificity(1), was used in a two-step procedure. Ovomucoid, a high-molecular-weightglycoprotein, was used as a second-step reagent due to its specific binding affinityfor WGA. The glycoprotein was complexed with colloidal gold at pH 5.4. Sec-tions 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 to60 min at room temperature in a moist chamber. After thorough washing withPBS, the sections were incubated with the ovomucoid-gold complex (1:60 inPBS-polyethylene glycol 20000). The sections were washed with PBS, rinsed withdistilled water, and contrasted with uranyl acetate and lead citrate. The speci-ficity of the labeling was assessed by (i) incubation with WGA to which N-N9-N0-triacetylchitotriose (1 mg/ml in PBS) had been added previously; (ii) incu-bation with WGA, then with uncomplexed ovomucoid, and finally with theovomucoid-gold complex; and (iii) direct incubation with the gold-complexedovomucoid with the lectin step omitted.

Quantification of labeling. The density of labeling obtained with the WGA-ovomucoid-gold complex was determined by counting the number of gold par-ticles per square micrometer. Areas were determined by the point counting

method of Weibel (34) by using negatives of electron micrographs projected ontoa lattice. The amount of labeling in a specified wall area (Sa) was estimated bycounting the number of gold particles (Ni) on a photographic enlargement. Thedensity of labeling (Ns) was calculated as follows: Ns 5 Ni/Sa, where Ns is thenumber of gold particles per unit surface.

Greenhouse experiments. Experiments were carried out in a sandy loam soilconsisting of 82% sand, 2% silt, 15% clay, and 0.4% organic matter (pH 7.4) andhaving a moisture-holding capacity of 12%. The temperature ranged from 27 to30°C. Daily irrigation was provided. Trichoderma sp. was added to the soil as awheat bran-peat mixture (0.5%, wt/wt). Chopped potato soil containing R. solaniwas 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-olddried synthetic medium agar culture. Cotton (Gossypium barbardenso L.) seed-lings were used in the experiments, which were performed in six replicates byusing plastic pots, each containing 0.5 kg of soil (10 plants/replicate). The com-plete experiment was repeated twice.

RESULTSOverexpression of ech42. Ech42 is thought to be a very im-

portant component of the T. harzianum chitinolytic system interms of mycoparasitism (3, 7, 21–23, 29). We transformedT. harzianum with plasmid pQuiA9 in order to generate strainscarrying multiple copies of ech42. Plasmid pHATa was co-transformed as a marker, and protoplasts were selected on thebasis of hygromycin resistance. Transformants were subjectedto two rounds of monosporic selection. A Southern analysis of12 independent transformants revealed that there was ectopicintegration of the construct, which left the original gene intact(data not shown). Six transformants carrying one to six extracopies of ech42 were selected and were designated the QLtransformants.

ech42 disruption. We transformed T. harzianum with plas-mid pQuiA2.3D, a derivative of pQuiA2.3 (3) in which a smallportion (228 bp) of the ech42 coding region was replaced by acassette conferring hygromycin resistance. Transformants wereselected on the basis of hygromycin resistance, and 3 of 70transformants yielded the product expected if homologous in-sertion of the disrupting construct had occurred (Fig. 1, lanes1 through 3). Two of these transformants (DQ-1 and DQ-2)(Fig. 1, lanes 2 and 3) appeared to be homogeneous, whereasthe third (DQ-3) (lane 1) apparently was chimeric and carriedboth disrupted and wild type nuclei. These three cultures weresubjected to a third round of monosporic culturing, and genedisruption was confirmed by Southern analysis following diges-tion of genomic DNA with SalI. A 2.3-kb band was expectedfrom the wild type and a 3.5-kb band was expected from thedisruptant when the cultures were hybridized with an internalfragment of the ech42 coding region as the probe. All threeputative disruptants had the expected 3.5-kb band (Fig. 2, lanes

FIG. 1. DNA analysis of the gene disruption candidates: PCR-amplifiedproducts. Samples (100 ng) of total DNA from different transformants weresubjected 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 thebands corresponding to wild type ech42 (WT) and disrupted ech42 (DQ) areindicated on the right.

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1 through 3) and were clearly different from the wild type (Fig.2, lane 4).

Chitinase expression in the transformed Trichoderma. Wetested five multicopy transformants and the three disruptantsto determine the levels of protein and chitinase activity. Strainswere grown by using chitin as the sole carbon source, andsamples were collected at the time when the highest level ofactivity was detected in the wild-type strain. The QL transfor-mants had chitinase activities that were up to 42 times higherthan the activity of the wild type (Table 1). The disruptantsexhibited virtually no activity.

Protein samples from the disruptants and three of the QLtransformants 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 ac-tivity than wild-type T. harzianum exhibited. For the QL trans-formants two bands appeared close together when a singleband was expected.

Protein samples were subjected to a Western blot analysis byusing a rabbit polyclonal antibody, and a single band at theexpected molecular weight was detected (Fig. 4); no proteinwas detected in the two disruptants analyzed (Fig. 4, lanes 5and 6). Densitometric analysis of the Western blot indicated

that the QL transformants (Fig. 4, lanes 1 through 3) secretedsix times as much chitinase as the wild type secreted (lane 4).

Quantification of chitin labeling of R. solani cell walls. Di-rect confrontation experiments performed with some of thetransgenic Trichoderma strains and R. solani were carried out,and the results were studied at the microscopic level. Thedamage to the R. solani cell walls caused by QL-9, DQ-1, andthe wild type was evaluated microscopically by labeling chitinwith the WGA-ovomucoid-colloidal gold complex (Fig. 5). Assoon as 4 h after the initial contact, slight degradation of theRhizoctonia cell wall by the wild type was observed (Fig. 5A).The ability of disruptant DQ-1 to attack the cell wall-boundchitin did not seem to be affected (Fig. 5B). In contrast, mark-edly greater cell wall alterations were observed with overpro-ducing clone QL-9 at the same time (Fig. 5C, arrowheads).These effects were distinct when the density of labeling was

FIG. 2. Southern analysis. Total DNA was extracted, digested with SalI, andprobed with a HindIII fragment from the ech42 coding region. The expected sizesof the bands corresponding to wild type ech42 (WT) and disrupted ech42 (DQ)are indicated on the left, and the positions of molecular size standards areindicated on the right. FIG. 3. Chitinase expression in transformed Trichoderma sp.: detection of

extracellular chitinolytic activity from T. harzianum transformed strains. Proteinsamples were subjected to SDS-PAGE and renatured, and the activity wasrevealed by using 4-methylumbelliferyl b-D-N,N9,N0-triacetylchitotriose as thesubstrate. 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 differentconstructs used to transform T. harzianum are shown at the top.

FIG. 4. Western blot of extracellular proteins from T. harzianum trans-formed 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 toSDS-PAGE and transferred onto a nylon membrane. An antibody against Ech42was used for immunodetection. Lane 1, QL-10; lane 2, QL-9; lane 3, QL-7; lane4, 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. har-zianum are shown at the top.

TABLE 1. Chitinase activities secreted by transformedand wild-type T. harzianum strains

StrainChitinase activity

(pmol of MU/min/mgof protein)a

IMI206040 ............................................................................. 27QL-5....................................................................................... 620QL-7....................................................................................... 1,200QL-9....................................................................................... 910QL-10..................................................................................... 570QL-11..................................................................................... 850DQ-1....................................................................................... 2.1DQ-2....................................................................................... 3.4DQ-3....................................................................................... 5.1

a The results shown are the results of a representative experiment. Similarresults were obtained in three independent experiments. MU, 4-methylumbel-liferone.

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FIG. 5. TEM micrographs of cultures containing either wild-type or transformed T. harzianum and R. solani. Chitin was labeled with the WGA-ovomucoid-goldcomplex. (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 indicatepoints where R. solani cell wall degradation is apparent.

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used as an indirect measurement of the chitin content of thecell wall (Table 2). By 16 h the plasma membrane appeared tobe altered and the cell wall was clearly degraded by all strains(Fig. 5D). Although at 4 h there was a tendency toward a lowerlevel of chitin labeling in the Rhizoctonia cell walls when theRhizoctonia cells were confronted with QL-9, this tendency wasstatistically significant only at 16 h. The particle counts in theDQ-1 micrographs were similar to the particle counts in thewild-type micrographs (Table 2). We measured ech42 geneexpression in wild-type and QL-9 strains grown on PDA aloneand in the presence of the pathogen. The basal level of geneexpression 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 tocontrol R. solani was 33%, and the disease incidence whenwild-type T. harzianum was used to control S. rolfsii was 25%;the values for DQ-1 were similar. QL-9 significantly reduceddisease incidence due to R. solani (14%) but not disease inci-dence due to S. rolfsii. Similar results were obtained with otherQL transformants (data not shown).

Growth curves. We grew wild-type T. harzianum in minimalmedium containing glycerol supplemented or not supplement-ed with chitin as an inducer of ech42 expression. Under theseconditions, ech42 expression was detected by 72 h and reacheda maximum level at 110 h; no ech42 expression was observedwhen T. harzianum was grown in the absence of chitin (datanot shown). Two QL transformants, the DQ-1 disruptant, andthe wild-type strain were cultured as described above, and theirgrowth curves were determined (Fig. 7). After 85 h in thechitin-containing medium, the QL transformants had gained

more weight than DQ-1 or the wild type, but the growth of theQL transformants declined faster later on. In every case theloss of biomass was accelerated in the presence of chitin. Thiseffect was even more pronounced with the two QL transfor-mants. In the case of the disruptant, the decline in growth wasslower. We also grew Trichoderma sp. on PDA plates and MMplates amended with either glucose or chitin. In all cases, theradial growth of the transformants was not significantly differ-ent from the radial growth of the wild type (data not shown).

DISCUSSION

Recently, it has been proposed that the complex chitinolyticsystem of T. harzianum is a key component in mycoparasitism(3, 7). In an attempt to determine the role of Ech42 in themycoparasitic process, we constructed transgenic Trichodermastrains carrying multiple copies of ech42 and deletion mutants.Expression of ech42 was directed by its own promoter, and weexpected that in most transformants, the gene would be regu-lated both in time and space as it is in the wild type, resultingin production of the enzyme where and when it is normallyrequired, albeit in larger quantities.

There was almost no chitinase activity in the gene disrup-tants, while the QL transformants exhibited much higher levelsof chitinase activity than the wild type exhibited (Table 1). Theactivity observed corresponded to two protein bands on SDS-PAGE gels, although both bands may have originated from asingle protein because the two activities increased in parallel inthe overexpressants and neither activity was detected in thedisruptants. These bands may correspond to two different con-formations of the same protein. When the protein sampleswere boiled in the presence of b-mercaptoethanol for theWestern blot analysis, a single protein band that reacted withthe anti-Ech42 antibodies was detected, supporting this hy-pothesis. Our observations differ from observations made in

FIG. 6. Plant disease control by wild-type and transformed T. harzianum ingreenhouse experiments. Columns for each series (R. solani, S. rolfsii) markedwith different letters are significantly different (P 5 0.05), as determined byDuncan’s multiple range test. Control, R. solani or S. rolfsii in soil; QL-9, patho-gen plus QL-9; DQ-1, pathogen plus DQ-1; WT, pathogen plus wild-type T. harzia-num.

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

TABLE 2. Densities of labeling obtained with theWGA-ovomucoid-gold complex for cell walls of

R. solani during interactions with wild-typeand transformed T. harzianum strains

Strain

Density of gold particles(no. of particles per mm2) aftera:

4 hb 16 h

Wild type 140 6 16c 81 6 12QL-9 98 6 12 43 6 18DQ-1 120 6 7 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.

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previous studies (15, 28), in which activity bands with differentmolecular weights were detected in other T. harzianum strains.In other studies we have observed that the strain used in ourwork secretes at least two exochitinases (unpublished observa-tions). However, the conditions used in our assays favoredEch42 activity.

Direct confrontation experiments performed with the Tricho-derma QL and DQ transformants and R. solani did not revealobvious differences in the capacities of these organisms to over-grow Rhizoctonia sp. compared to the nontransformed strain invitro, except for slightly wider areas of apparent lysis in thecontact lines when the multicopy transformants were used(data not shown). Electron microscope investigations of thedamage to Rhizoctonia cell walls caused by DQ-1 and the wildtype revealed no significant differences in the chitin content.However, there were significant differences between QL-9 andthe wild type. These differences were not proportional to thedifferences in chitinase activity and supported the hypothesisthat the combined action of a set of chitinases is required forefficient degradation of chitin in the fungal cell wall. These re-sults also suggest that in the complex chitinolytic system spe-cific enzymatic activities are redundant. In all cases major al-terations, including retraction of the plasma membrane andaggregation of the cytoplasm, occurred prior to cell wall deg-radation, suggesting that an alternative mechanism may be theprimary determinant in the mycoparasitic process of the Tricho-derma strains analyzed.

We were surprised that there was no significant differencebetween the disruptants and the wild type in the greenhousebiocontrol tests (Fig. 6). An analogous situation has been de-scribed for the plant-pathogenic fungus Cochliobolus carbo-num, in which disruption of an endopolygalacturonase genedid not affect pathogenicity (30). One possible explanation isthat other enzymes of the chitinolytic system of Trichodermasp. are sufficient for control of both R. solani and S. rolfsii. Inaddition, it has been shown that the lack of a certain proteincan be compensated for by altering the levels of other proteinswith similar activities (31).

Treatment with the transgenic Trichoderma strain that pro-duced the highest chitinase levels in vitro reduced the diseaseincidence 9 to 19% compared to treatment with the wild type(Fig. 6). In contrast, we previously observed a fivefold reduc-tion in the incidence of disease caused by R. solani when weused a transgenic Trichoderma strain carrying multiple copiesof the mycoparasitism-related gene prb1 (10). We suggest thatthe levels of chitinases naturally secreted by Trichoderma spe-cies are high enough for efficient biocontrol of the phytopatho-genic fungi tested. Thus, an increase in the production of oneof the chitinases would not dramatically affect the mycopara-sitic capacity.

Taken together, our data demonstrate that expression ofech42 is one part of a series of responses of Trichodermaspecies to the presence of a potential host and that this gene isnot essential for effective plant disease control.

ACKNOWLEDGMENTS

We thank Jose Ruız-Herrera, Luis Herrera-Estrella, and BenjaminHorwitz for critically reading the manuscript. We thank G. E. Harmanfor donating the anti-Ech42 antibodies.

This work was supported in part by grant 3527P-N9607 fromCONACYT and by grant C/2446-1 from IFS to A.H.-E, by a grantfrom the German-Israel Foundation to I.C., and by CONACYT doc-toral fellowships to C. Carsolio and C. Cortes.

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