INTRODUCTION

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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 -(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 -(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, 21-23, 29). The corresponding gene (ech42) is strongly induced during fungus-fungus 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

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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 pHAT, 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 EMBL3 (3) was subcloned into pBluescript, generating plasmid pQuiA9. The inserted fragment contained the entire coding region for Ech42 plus approximately 2 kb of the 3' region and 5 kb of the 5' region. Plasmid pQuiA9 was used to transform T. harzianum protoplasts as previously described (16, 20).

PCR amplification. PCR were carried out with T. harzianum genomic DNA from the different transformants obtained with plasmid pQuiA2.3. Primers were designed so that homologous recombination or ectopic integration resulted in different band patterns. Briefly, the forward primer (5' GGACCAGGTGCTGTT 3') hybridized on the 5' side of the gene (ech42) but outside the fragment contained in pQuiA2.3, and the reverse primer (5' TAGTTGAGACCGCTTCG 3') hybridized in the coding region downstream of the insertion site of the cassette. Taq polymerase (Gibco-BRL) was used for all amplifications. The following PCR program was used: a hot start consisting of 5 min at 95°C; 25 cycles consisting of 1 min at 94°C, 2 min at 60°C, and 3 min at 72°C; and a final extension period consisting of 7 min at 72°C. 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 70°C until they were used.

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 4°C, frozen, and lyophilized again. The samples were resuspended in 300 to 500 µl of H₂O, divided into aliquots, frozen, and kept at 70°C until they were used. One to ten micrograms of each sample was resuspended in Laemmli buffer (19) without -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 casein-EDTA 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 µg of 4-methylumbelliferyl -D-N,N',N"-triacetylchitotriose and incubated at 37°C 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 -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 25°C 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 4°C and postfixed with 1% (wt/vol) osmium tetroxide in the same buffer for 1 h at 4°C. 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 -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 µg/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-N'-N"-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 WGA-ovomucoid-gold complex was determined by counting the number of gold particles per square micrometer. Areas were determined by the point counting method of Weibel (34) by using negatives of electron micrographs projected onto a lattice. The amount of labeling in a specified wall area (S_a) was estimated by counting the number of gold particles (N_i) on a photographic enlargement. The density of labeling (N_s) was calculated as follows: N_s = N_i/S_a, where N_s 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 30°C. 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

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Overexpression of ech42. Ech42 is thought to be a very important component of the T. harzianum chitinolytic system in terms of mycoparasitism (3, 7, 21-23, 29). We transformed T. harzianum with plasmid pQuiA9 in order to generate strains carrying multiple copies of ech42. Plasmid pHAT 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 pQuiA2.3, 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 (Q-1 and Q-2) (Fig. 1, lanes 2 and 3) appeared to be homogeneous, whereas the third (Q-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 1 through 3) and were clearly different from the wild type (Fig. 2, lane 4).

View larger version (89K): [in this window] [in a new window]   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, Q-3; lane 2, Q-1; lane 3, Q-2; lane 4, another Q 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 (Q) are indicated on the right.

View larger version (80K): [in this window] [in a new window]   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 (Q) are indicated on the left, and the positions of molecular size standards are indicated on the right.

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.

View larger version (70K): [in this window] [in a new window]   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 -D-N,N',N"-triacetylchitotriose as the substrate. Lane 1, Q-1; lane 2, Q-2; lane 3, Q-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.

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Western blot of extracellular proteins from T. harzianum transformed strains. A 2.5-µg 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, Q-2; lane 6, Q-1; lane 7, recombinant Ech42 from E. coli. Schematic representations of the different constructs used to transform T. harzianum are shown at the top.

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, Q-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 Q-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 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 Q-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).

View larger version (139K): [in this window] [in a new window]   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) Q-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.

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 Q-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).

View larger version (49K): [in this window] [in a new window]   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 = 0.05), as determined by Duncan's multiple range test. Control, R. solani or S. rolfsii in soil; QL-9, pathogen plus QL-9; Q-1, pathogen plus Q-1; WT, pathogen plus wild-type T. harzianum.

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 Q-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 more weight than Q-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).

View larger version (12K): [in this window] [in a new window]   FIG. 7.   Growth curves for transformed T. harzianum strains. Strains were grown in MM containing glycerol () or MM containing glycerol and chitin ().

	DISCUSSION

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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 SDS-PAGE 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 -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 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 Q 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 Q-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.