Biol Res 35: 401-410, 2002

The expression of extracellular fungal cell wall
hydrolytic enzymes in different Trichoderma harzianum
isolates correlates with their ability to control
Pyrenochaeta lycopersici.

LUZ MARÍA PÉREZ ^{** 1}, XIMENA BESOAÍN², MAURICIO REYES^1,3, GONZALO PARDO²
AND JAIME MONTEALEGRE⁴.

¹ Laboratorio de Bioquímica, Facultad Ciencias de la Salud, Universidad Nacional Andrés Bello.
² Facultad de Agronomía, Universidad Católica de Valparaíso.
³ Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas,
Universidad de Chile
⁴ Departamento de Sanidad Vegetal, Facultad de Ciencias Agronómicas, Universidad de Chile.

ABSTRACT

Four isolates of Trichoderma harzianum (ThN3, Th11, Th12 and Th16) were selected for their ability to control the in vitro development of the tomato root pathogen Pyrenochaeta lycopersici. Analysis of the mechanisms involved in biocontrol showed that the formation of non-volatile metabolites appears to be one of those involved in biocontrol of P. lycopersici by all T. harzianum isolates tested. Nevertheless, the higher secretion of chitinases, both in number of isoenzymes and activity by the Th11 strain, correlated well with its higher ability to control this agent in laboratory and greenhouse experiments as compared to the other T. harzianum isolates tested. The secretion of ß -1,3-endoglucanases and/or proteases appeared to have less significance than endochitinases in the biological control of P. lycopersici.

Key terms: biological control, corky root, tomato root diseases, Trichoderma harzianum, Pyrenochaeta lycopersici.

INTRODUCTION

Pyrenochaeta lycopersici causes the corky root disease in tomato, in the main area of tomato production under green house conditions in Chile. During the winter season, P. lycopersici is found to be the main pathogen (Araya 1994). Its control is presently accomplished through fumigation of soils with methyl bromide (Campbell et al, 1982). However, although methylbromide is very effective in controlling this agent, it is also highly toxic, contributes to the enlargement of the ozone hole and to environmental pollution, and its use will soon be banned (Ristaíno and Thomas 1997).

An alternative to decreasing the use of chemicals for controlling plant pathogens is to replace them, at least in part, with antagonistic microorganisms. Fungi from the Trichoderma genus are well known as biocontrol agents, especially T. harzianum (Harman and Kubicek, 1998; Vannacci and Gullino 2000). Nevertheless, it appears that each Trichoderma isolate behaves differently when faced with the same plant pathogen. The differences observed in the biocontrol activity could depend on the ability of each isolate to produce antibiotics (Dennis and Webster 1971a; b) and/or to express genes that codify for extracellular fungal cell wall hydrolyzing enzymes such as chitinases, ß -1,3-glucanases and/or proteases, or in the specific isoenzyme pattern expressed by each isolate (Grondona et al, 1997).

This research therefore sought to characterize several local Trichoderma harzianum isolates on the basis of their ability to secrete fungal cell wall hydrolyzing enzymes (including the corresponding isoenzyme pattern) and to form antibiotics, along with the development of biocontrol assays on P. lycopersici using the Trichoderma isolates already characterized. Our aim was to establish whether there was a correlation between the molecular characteristics of the T. harzianum isolates used and their behavior as biocontrol agents of P. lycopersici. This information is essential for further selection of the Trichoderma isolate with the best characteristics to be used as a biocontrol agent of P. lycopersici at the field level.

MATERIALS AND METHODS

Trichoderma isolation

Trichoderma were isolated either from tomato monoculture suppressive soils (Th11, Th12 and Th16) or from garden soils (ThN3). They were cultured on potato-dextrose-agar, PDA (DIFCO), until pure cultures were obtained. The four isolates were identified as Trichoderma harzianum, based on their morphology after cultivation in PDA, malt-agar and Czapek agar (Pérez et al, 1991).

Tomato pathogen

P. lycopersici was isolated from roots of tomato plants showing symptoms of corky root. The pathogen was identified according to Clerjeau (1976).

INHIBITORY ASSAYS

Dual cultures

An 8-mm disk of a pure P. lycopersici culture was seeded on one side of a Petri dish containing PDA and cultured for seven days at 15° C. The growth of the pathogen was then checked, and the plate was inoculated with another 8-mm disk of a pure culture of one of the T. harzianum isolates (ThN3, Th11, Th12 or Th16), placed on the site opposite the P. lycopersici. The plates were cultured for another 96 h at 15° C. The growth diameter of the pathogen was measured and compared to the growth of the pathogen at the time that Trichoderma was inoculated on the plate. Controls were run on plates inoculated with P. lycopersici and mock inoculated with an 8-mm disk of PDA. Each experiment considering a single Trichoderma isolate was run in triplicate and repeated at least three times. Results are expressed as means +S.D. in cm of the P. lycopersici growth in the presence and absence of any T. harzianum isolate. The Student t test was performed at p <0.05.

Production of volatile inhibitory
metabolites

The centers of half Petri dishes containing PDA were seeded with an 8-mm disk of pure cultures of either P. lycopersici or one of the T. harzianum isolates (ThN3, Th11, Th12 or Th16). One half-plate containing a recently-seeded T. harzianum isolate and another containing P. lycopersici seeded seven days earlier were placed face-to-face preventing any physical contact between fungi, and the growth of P. lycopersici was measured at this time. The half-plates were sealed to isolate the interior atmosphere and to prevent the loss of any volatiles that formed. The plates were incubated at 15°C for another 96 h, and the growth of the pathogen was measured and compared to controls developed in the absence of the bioantagonist (mock inoculation with an 8-mm disk of PDA). Each experiment considering a single Trichoderma isolate was run in triplicate and repeated at least three times. Results are expressed as means + S.D. in cm of the growth of P. lycopersici in the presence and absence of any T. harzianum isolate. The Student t test was performed at p <0.05.

Production of diffusible inhibitory
metabolites

The centers of PDA plates covered with a dialysis membrane were inoculated with an 8-mm disk of each of the antagonistic Trichoderma. After incubation for 72 h at 28°C, the membrane with the grown Trichoderma isolate was removed, and the center of the plate was inoculated with an 8-mm disk of a pure P. lycopersici culture. The plates were further incubated at 15°C for 96 h, at which time the pathogen growth was measured. Controls were run with mock inoculated plates on the dialysis membrane and inoculated with P. lycopersici. Each experiment considering a single Trichoderma isolate was run in triplicates and repeated at least three times. Results are expressed as means + S.D. in cm of growth of P. lycopersici in the presence and absence of any T. harzianum isolate. The Student t test was performed at p <0.05.

Submerged cultures of Trichoderma and
quantitation of enzyme activities

Each Trichoderma isolate was grown in liquid Mandels medium (Mandels et al, 1974) using 4g per liter of either crab chitin, yeast glucans, or cell walls from P. lycopersici as the sole carbon source. Supernatants from 8-day cultures were used for the analysis of chitinases, ß 1,3-glucanases and proteases after native electrophoresis of proteins (Pan et al, 1991).

Greenhouse experiments

Experiments were run in the greenhouse with winter-simulated conditions, where soil temperatures fluctuated between 10° C and 20° C. Drip irrigation was used and fertilizers were applied during watering. Tomato seedlings (Fortaleza cv.) were grown in `speedlings' until two true leaves developed, whereupon they were transferred to pots containing 3L of naturally infected soil plus different treatments, as follows:

The different T. harzianum isolates were applied as alginate beads prepared as described by Montealegre and Larenas (1995). They were placed in direct contact with seedling roots at the moment of seedling transfer. The dry weight of aerial and root portions of the plants, as well as root damage were evaluated. Damage was evaluated using the following scale: 0, healthy roots; 1, one to five % lesions; 2, six to 15% lesions; 3, 16 to 30% lesions; 4, 31 to 60% lesions, and 5, 61 to 100% lesions (Pardo 1999). A complete randomized experimental design was done using 8 replicates per treatment, considering that the experimental unit consisted of one plant contained in one pot. The results were analyzed by using ANOVA, and the means were compared using the Tukey test with 5% significance.

RESULTS AND DISCUSSION

Antagonistic effect of Trichoderma isolates
on P. lycopersici

All four Trichoderma isolates (ThN3, Th11, Th12 and Th16) could control the development of P. lycopersici in dual culture in vitro experiments (Table I). Nevertheless, none of the isolates appeared to be more effective than the others in reducing the growth diameter of the pathogen isolate. When biocontrol was analyzed in terms of the formation of volatile and non-volatile metabolites that could affect fungal growth, a decrease could not be observed in the development of P. lycopersici due to the formation of volatile metabolites (Table I). These results suggest that these types of compounds are not involved in the control of the development of P. lycopersici by any of the T. harzianum isolates tested. It appears that the lack of effect in controlling P. lycopersici development is not due to a lack of ability of these biocontrol agents to produce volatile metabolites, as the inhibitory effect of these compounds on Phytophthora parasitica (Besoaín et al, 2001) has been demonstrated. On the other hand, the formation of non-volatile metabolites by any of the Trichoderma isolates tested resulted in a significant decrease of fungal growth (Table I). These non-volatile metabolites could correspond to antibiotic peptaibols formed by T. harzianum (Schirmböck et al, 1994), or to diffusible factors ( Cortés et al, 1998) whose molecular weight allow them to cross the dialysis membrane into the culture medium. However, the production of non-volatile antibiotics in all four isolates apparently is not the only mechanism used to control P. lycopersici development by any of the T. harzianum isolates tested. In fact, the decrease in fungal growth in these experiments was less effective than the one observed in dual culture assays (Table I), suggesting that an additional biocontrol mechanism could be involved in controlling P. lycopersici. This could correspond to a differential expression of extracellular fungal cell wall hydrolyzing enzymes. The participation of these two mechanisms, i.e.: the production of non-volatile metabolites and extracellular hydrolytic enzymes, has been demonstrated for other T. harzianum isolates controlling other phytopathogenic fungi (Schirmböck et al, 1994).

Secretion of fungal cell wall hydrolytic
enzymes by T. harzianum isolates ThN3,
Th11, Th12 and Th16.

Endochitinases.

The number of isoenzymes with endochitinase activity secreted into a liquid medium was characteristic of each T. harzianum isolate and depended up the carbon source provided (crab chitin or P. lycopersici cell walls). No endochitinase activity was detected on basic gels when supernatants from ThN3 grown in the presence of P. lycopersici cell walls were analyzed (Fig. 1). Nevertheless, this same isolate secreted one negatively-charged endochitinase when crab chitin was used as the sole carbon source (Fig. 1), suggesting that ThN3 is unable to induce this enzyme specifically in response to the presence of cell walls of the pathogen, although it is able to secrete endochitinases in the presence of cell walls from Fusarium solani (Reyes et al, 2000; Pérez et al, 2001). Th11 secreted three different negatively-charged endochitinases in the presence of crab chitin and five isoenzymes in the presence of cell walls from the phytopathogen (Fig. 1). Th12 and Th16 secreted one negatively-charged endochitinase with identical electrophoretic migration, when grown in the presence of crab chitin or of cell walls from P. lycopersici. The intensity of this single band of activity slightly increased when these biocontrol fungi were grown in the presence of cell walls from P. lycopersici. Therefore, each T. harzianum shows a different ability to secrete endochitinases, which also depends on the carbon source used in the culture medium.

Figure 1: Extracellular endochitinase activity of different isolates of T. harzianum grown in crab chitin (a) or in P. lycopersici cell walls (b) as the sole carbon source. Native PAGE at pH 8.8 separated proteins from supernatants of culture media. Endochitinase activity was visualized after incubating the polyacrylamide gel with an auxiliary 2% (w:v) agarose gel containing glycol chitin and further incubation with 0.01% (w:v) fluorescent brightener 28. Arrows show endochitinase activity.

When analyses were done in acid gels, positively charged endochitinases at pH 4.4. were visualized. In these conditions, no endochitinase activity was found in supernatants of ThN3 cultured either in crab chitin or in cell walls from P. lycopersici as the sole carbon source (Fig. 2). On the other hand, supernatants from Th11 showed three bands of endochitinase activity independently of the culture medium (Fig. 2). However, the intensity of bands of enzymatic activity decreased in supernatants from cultures in the presence of pathogen. Finally, Th12 and Th16 showed one endochitinase band of activity when grown in crab chitin as the sole carbon source, but only Th12 had the ability to maintain the expression of this extracellular endochitinase activity in the presence of P. lycopersici cell walls. In these assay conditions, Th11 once again demonstrated the ability to secrete the highest number of isoenzymes of endochitinase when grown in the presence of cell walls from the phytopathogen.

The analysis of endochitinases both in basic and acidic conditions allows us to conclude that each T. harzianum isolate has a different ability to induce the secretion of this enzyme activity and that it also depends on the chitin-derived carbon source. These results are consistent with the genetic diversity described for different isolates of the fungus (Gómez et al, 1997) and thus with the presence of one or more genes that code for extracellular endochitinases, which could be induced by the chitin components of the cell wall of P. lycopersici. Within this context, Th11 is the isolate that shows the greatest ability to secrete more endochitinases (acidic plus basic) in the presence of cell walls from P. lycopersici as compared to ThN3, Th12 or Th16.

Figure 2: Extracellular endochitinase activity of different isolates of T. harzianum grown in crab chitin (a) or in P. lycopersici cell walls (b) as the sole carbon source. Native PAGE at pH 4.4 separated proteins from supernatants of culture media. Endochitinase activity was visualized after incubating the polyacrylamide gel with an auxiliary 2% (w:v) agarose gel containing glycol chitin, and further incubation with 0.01% (w:v) fluorescent brightener 28. Arrows show endochitinase activity.

ß -1,3-glucanases

ß -1,3-glucanases are enzymes that are also involved in biocontrol activity of several microorganisms (Vásquez-Garcidueñas et al, 1998). Analysis of this activity in basic gels using supernatants from the ThN3 isolate grown in yeast glucans showed the presence of only one negatively-charged ß -1,3-glucanase (Fig 3A). A second band of ß -1,3-glucanase activity was observed in basic gels after growth of the biocontroller in the presence of P. lycopersici (Figure 3A). Th11 expressed two extracellular ß 1,3glucanase isoenzymes, and a third band of activity was observed after growth in the presence of walls of the phytopathogen. Migration of this latter band was higher than the second band observed for ThN3, suggesting that the third protein expressed by Th11 has a more basic isoelectric point (Fig. 3A). Th12 secreted one ß 1,3glucanase in the presence of yeast glucans and one more isoenzyme in the presence of cell walls from P. lycopersici. Finally, Th16 secreted two ß 1,3glucanase isoenzymes, both in the presence of yeast glucans or P. lycopersici cell walls (Fig. 3A).

When analyses were done on acidic gels (Fig 3B), it could be observed that ThN3 and Th11 were able to secrete up to four positively charged ß 1,3glucanases when grown in the presence of yeast glucans. Migration of ß 1,3glucanases was coincident in these two isolates. In the presence of cell walls from P. lycopersici, only Th11 had the ability to maintain the expression of extracellular ß 1,3glucanase activity, although only the two isoenzymes with highest migration could be visualized (Fig 3B). Th12 and Th16 expressed two and three ß 1,3glucanase isoenzymes, respectively, in the presence of P. lycopersici, but only Th16 had the ability to express extracellular ß 1,3glucanase activity when grown in the presence of yeast glucans (Fig 3B). Again, these results are consistent with the genetic diversity shown by T. harzianum (Gómez et al, 1997), and agree with the ability of the isolates to secrete different amounts and levels of ß-1,3-endoglucanases. As opposed to the secretion of endochitinases, the Th11 isolate does not appear to be the one that shows the best secretion of these enzymes in the presence of cell walls from P. lycopersici because Th12 showed the secretion of the same number of ß 1,3glucanases and Th16 expressed a third extracellular ß 1,3glucanase. Nevertheless, the specific isoenzyme pattern of each T. Harzianum isolate could contribute, at least in part, to their biocontrol activity.

Figure 3: Extracellular ß-1,3-glucanase activity of different isolates of T. harzianum grown in yeast glucans (a) or in P. lycopersici cell walls (b) as the sole carbon source. Native PAGE at pH 8.8 (A) and pH 4.4 (B) separated proteins from supernatants of culture media. ß-1,3-glucanase activity was visualized after incubating the gel with 1% (w:v) laminarin and developing bands of activity with 0.15 % (w:v) triphenyltetrazolium. Arrows show ß-1,3-glucanase activity.

Proteases

Analysis of proteases showed that all isolates had the ability to secrete this enzyme activity. As opposed to endochitinases or ß 1,3glucanases, only zones of degradation of the hemoglobin contained in the gel were observed, suggesting that these enzymes were active even during development of electrophoresis (Fig 4). The fact that proteolytic activity was observed in supernatant from all four T. harzianum isolates grown in the presence of crab chitin suggests that constitutive extracellular activity is expressed in all four biocontrollers.

The constitutive extracellular protease activity increased after development of each T. harzianum isolate in the presence of P. lycopersici cell walls, with the exception of ThN3, where enzyme activity was lower than that observed for this isolate grown in the presence of the phytopathogen (Fig 4). It is interesting to mention that discrete protein bands were observed after the development of protease activity, that is, some of the fungal proteins were not degraded by the proteases secreted with them These results suggest that they could correspond to a protein resistant to proteolysis and whose activity may be important for biocontrol.

It may be concluded f the characterization of the ThN3, Th11, Th12 and Th16 isolates that although they belong to the same species, they show different properties related to the expression of extracellular fungal cell wall hydrolytic enzymes when grown in the presence of P. lycopersici cell walls, confirming their genetic variability (Gómez et al, 1997). The fact that isolate Th11 expresses the largest number of isoenzymes of chitinases as well as a high proteolytic activity in the presence of cell walls of P. lycopersici, suggests that these characteristics could be considered when analyzing biocontrol experiments. The number of chitinase isoenzymes as well as a high proteolytic activity has been described as relevant for biocontrol activity in other T. harzianum isolates (Grondona et al, 1997). In order to test this fact, greenhouse experiments were conducted using those Trichoderma isolates that expressed extracellular chitinases and increased their proteolytic activity when grown in the presence of P. lycopersici isolated cell walls: Th11, Th12 and Th16.

Figure 4: Extracellular protease activity of different isolates of T. harzianum grown in crab chitin (a) or in P. lycopersici cell walls (b) as the sole carbon source. Native PAGE at pH 4.4 in hemoglobin containing gels, separated proteins from supernatants of culture media. Protease activity was visualized after incubating the gel with Coomassie blue. Clear zones on the gel indicate proteolytic activity

Greenhouse experiments

The dry weight of the aerial portion of tomato plants treated with Th11, Th12 or Th16, showed that Th11 at a dose of 2g per plant and Th12 at a dose of 4g per plant were significantly increased when compared to control plants (Table II). On the other hand, the same isolates at the same doses behaved similarly to methylbromide when their roots were analyzed (Table II). These results correlate with the ability of Th11 to induce more chitinases than Th12, and with a higher proteolytic activity of Th12 over Th11, which shows a similar effect at half the dose. Treatment of tomato plants with Th11 at a dose of 4 g per plant did not improve the effect observed at half this dose; in fact, the plant behavior was similar to controls, suggesting either a deleterious effect due to internal competence of Trichoderma or a phytotoxic effect (Hoitink and Boehm, 1999).

Again, a clear reduction in lesions was obtained after treating plants with a dose of 2 g per plant of Th11 (Table II), this treatment, along with that of methylbromide, being the only one that differed from control. The use of Th11 at a dose of 4 g per plant, or of Th12 or of Th16 at both 2 and 4 g per plant did not result in an effective control of lesions produced by P. lycopersici. This may be explained by the fact that Th12 and Th16 express fewer chitinase isoenzymes than Th11 and that as a consequence of competition resulting from a high concentration of the biocontroller, Th11 could also show a phytotoxic effect on the plant. (Hoitink and Boehm, 1999).

The results of the greenhouse experiments clearly agreed with Campbell et al (1982) that methylbromide was the best treatment for controlling P. lycopersici, but they also showed that Th11 can control, at least in part, the disease caused by P. lycopersici. It appears that endochitinase activity, represented by several isoenzymes in Th11, is one of the primary mechanisms used by this isolate in its biocontrol activity. Nevertheless, extracellular proteolytic activity also contributes to biocontrol because Th12, at least at a dose of 4g per plant, had an effect similar to Th11 at 2g per plant. In this context, the expression of extracellular ß-1,3-glucanases appears to be unimportant for biocontrol of P. lycopersici by any of the T. harzianum isolates tested. In fact, treatments with any dose of Th16, which showed the highest number of these isoenzymes as well as a high proteolytic activity, were similar to controls. In conclusion, Th11 shows advantages over the other T. harzianum isolates tested in terms of expression of extracellular fungal cell wall hydrolyzing enzymes and of biocontrol activity through production of non-volatiles which agree with results obtained in greenhouse experiments. Therefore, Th11 could be selected as a promising isolate for its use as an alternative biocontroller of P. lycopersici.

ACKNOWLEDGEMENTS

The present work was funded by grants from FONDECYT (1990785) and UNAB (DI 77-00). We also acknowledge Dr. Eduardo Piontelli (University of Valparaíso) for his help in fungal identification.

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Corresponding author: Luz M. Pérez. Laboratorio de Bioquímica, Facultad Ciencias de la Salud, Universidad Andrés Bello. Sazie 2325, Santiago de Chile. Telephone: 56-2-6618411. Fax: 56-2-6618390. e-mail: lperez@abello.unab.cl

Received: March 11, 2002. In revised form: July 25, 2002. Accepted: July 29, 2002