Electrophoretic characterization of chitinases as a tool for the identification of Trichoderma harzianum strains

Mycol. Res. 102 (3) : 373–377 (1998)

373

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Electrophoretic characterization of chitinases as a tool for the identification of Trichoderma harzianum strains

H E D V A S C H I C K L ER1, B A T C H E N D A N I N - G E H A L I2, S H O S H A N H A R AN2 A N D I L A N C H E T2 " The Kennedy-Leigh Center for Horticultural Research # Otto Warburg Center for Agricultural Biotechnology, the Hebrew University of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences, Rehovot 76100, Israel

The identification of Trichoderma strains is important for their application as biocontrol agents. We used a two-dimensional analysis, in which extracellular proteins of Trichoderma harzianum strains T-35, Y and TM were first separated according to their isoelectric point and then according to their molecular mass. Chitinase activities were detected in situ after the second separation. Each of the three strains exhibited a unique pattern of 3–5 different chitinases (1 or 2 N-acetyl-β-glucosaminidases, and 2 or 4 endochitinases). These unique profiles can be used to differentiate among strains within this species. The suggested procedure makes use of a property of T. harzianum that is directly involved in the mycoparasitic interaction, making it suitable for specific biocontrol applications.

Trichoderma harzianum Rifai is a mycoparasite which is known as a biocontrol agent against several plant pathogens (Chet, 1987). Chitin is a major cell-wall component of most phytopathogenic fungi, and chitinases have been found to be directly involved in the mycoparasitic interaction between Trichoderma and its host fungus (Papavizas, 1985 ; Chet, 1987 ; Chet, Barak & Oppenheim, 1993 ; Haran et al., 1996). Along with other lytic enzymes such as glucanases and proteases, chitinases are excreted to degrade the pathogen’s cell walls, and this capability is suggested to be one of the main mechanisms for the biocontrol efficacy of Trichoderma (Chet, 1987). The chitinolytic system of T. harzianum is composed of six chitinases, each with its specific mode of hydrolysing chitin (Haran et al., 1995). Strains within T. harzianum differ with respect to their biocontrol effectiveness against phytopathogenic fungi (Elad, Chet & Henis, 1982 ; Sivan & Chet, 1989). An important step in utilizing the full potential of this fungus as a biocontrol agent is, therefore, the ability to identify specific strains within the species classification. The only comprehensive taxonomic monograph for Trichoderma was produced by Rifai (1969), who classified Trichoderma into nine species aggregates, based on morphological descriptions of colony growth and conidiophores. In a recent review, Samuels (1996) emphasized that this type of classification is often a source of confusion, because of significant variations which need to be defined for each group, and presented the more recent macromolecular approach to Trichoderma taxonomy. Characters derived from nucleic acids and enzymes are attractive because they seem to offer the possibility of greater objectivity than do the traditionally observed and analysed data. A classification based on enzyme variation in electrophoretically detectable proteins has been

suggested by several authors (reviewed by Samuels, 1996). In this work we improved this identification system by using a more accurate two-dimensional gel analysis to characterize the chitinolytic system, which is directly related to the biocontrol efficacy of Trichoderma strains.

MATERIALS AND METHODS Cultures and protein extraction T. harzianum strain TM was isolated from Mexican soil, and kindly provided by Dr A. Herrera-Estrella. Strains Y and T-35 (ATCC 20691) were isolated in Israel. The strains were grown on potato dextrose agar (PDA) (Difco, Detroit, MI). Twenty mycelial discs (6 mm diam.) from 72 h old PDA cultures were placed in 250 ml flasks containing 100 ml synthetic medium (SM), consisting of (l−") : 15 g glucose, 2 g MgSO \7H O, % # 0±6 g K HPO , 0±15 g KCl, 1 g NH NO , and the following # % % $ trace elements : 0±005 g FeSO \7H O, 0±006 g MnSO \H O, % # % # and 0±004 g ZnSO \H O (Okon, Chet & Henis, 1973). The % # flasks were shaken at 30 °C, 110 rpm for 4 d. This inoculum was then blended using a Janke & Kunk (Germany) ultraturrax. Aliquots (7 ml) of this homogenate were transferred to 250 ml flasks, each containing 100 ml SM supplemented with 3 g collodial chitin as the sole carbon source. The flasks were shaken at 110 rpm for 48 h at 30°. Protein extracts were concentrated from T. harzianum growth media to recover their enzymic activities. Extracellular proteins were isolated as follows : the growth medium was filtered through mesh-cloth (120 mm), and 4 µ leupeptine and 0±2 m phenylmethylsulphonyl fluoride (PMSF) (both from Sigma Chemical Co., St Louis, MO) were added. The

Trichoderma identification by chitinase profiles Strain T-35

1

2

3

374 Protein concentration was determined according to Bradford (1976) using Bio-Rad (Hercules, CA) protein-assay dye reagent and bovine serum albumin as the protein standard.

4

M (kDa) 106 80

CHIT102

49

CHIT52 CHIT42

32

CHIT33 CHIT31

CHIT73

27

Strain Y

1

2

3

4

M (kDa) 143 97

CHIT102 CHIT73 CHIT52 CHIT42

50

Determination of isoelectric point (pI ) A solution consisting of 300 µg extracellular protein, 2 % ampholytes 3}10 (Sigma) and distilled water (50 ml total volume) was loaded onto a Rotofor-cell (Bio-Rad). The Rotofor system fractionates complex protein samples in free solution using preparative isoelectric focusing. The ampholytes create a linear pH gradient, in which proteins migrate until the net charge on the molecule is zero. In this way the proteins become focused at their individual pIs. Purified proteins are harvested in free solution within specific pH intervals, and are usually found in three adjacent fractions with the highest concentration in the middle fraction. The pH in each fraction was measured using a pH meter (Radiometer, Copenhagen), and chitinase activity was detected as follows. Identification of enzymic activities

35 CHIT33 CHIT31

30

Strain TM

1

2

3

4

M (kDa) 143

CHIT102

97 50 35

CHIT42 CHIT33

30

Fig. 1. Detection of chitinolytic activities of proteins produced by T. harzianum grown on chitin as the sole carbon source. Lanes contain 25 µg of extracellular protein, renatured following their separation by SDS–PAGE. Temperature treatments prior to loading were : lanes 1 and 3, room temperature ; lanes 2 and 4, 3 min at 55°. Representative gels from 2–4 runs are shown. Lanes 1 and 2 : detection of chitinolytic activity using 4-methylumbelliferyl-N-acetyl-β-glucosaminide (4-MU-GlcNAc) as the substrate. Lanes 3 and 4 : detection of chitinolytic activity using 4-methylumbelliferyl-β-N,N«-diacetylchitobioside [4-MU-(GlcNAc) ] as the substrate. Mol# ecular masses of the Trichoderma enzymes were estimated from the regression equation : log molecular mass of protein standards v. distance migrated. Enzymes were designated according to their molecular mass.

filtrate was then dialysed and concentrated in a MicroProDiCon membrane (MWCO : 25 000) against distilled water at 4° in a Micro-ProDiCon negative-pressure micro-protein dialysis}concentrator, as described by the manufacturer (Spectrum Medical Industries Inc., Houston, TX).

Enzymic activity was first identified in solution using, separately, nitrophenyl N-acetyl-β--glucosaminide [pnp(GlcNAc)] and nitrophenyl-β--N,N«-diacetylchitobioside [pnp-(GlcNAc) ] as enzyme substrates. Activity was assayed # by monitoring the rate of formation of p-nitrophenyl from the substrates (Roberts & Selitrennikoff, 1988). Fractions in which activity was detected were loaded onto gels (at a volume equivalent to 0±25 OD h−") for second-dimensional analysis and the various chitinase activities were detected as previously described by Haran et al. (1996) : proteins were prepared in Laemmli buffer (Laemmli, 1970) without 2-mercaptoethanol. The denatured protein extracts were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) in 1±5 mm gels with 4 % acrylamide (stacking gel) and 10 % acrylamide (separating gel), in a Mighty Small II protein electrophoresis cell (Hoefer Scientific Instruments, San Francisco, CA). Enzymes were reactivated by removing SDS, following the casein}EDTA procedure (McGrew & Green, 1990) modified by Haran et al. (1995). Chitinolytic enzymes were detected in situ using two highly sensitive substrates that produce a fluorescent product upon enzymic hydrolysis : (a) 4-methylumbelliferyl-N-acetyl-β-glucosaminide (4-MU-GlcNAc) ; (b) 4-methylumbelliferyl-β-N,N«-diacetylchitobioside [4-MU-(GlcNAc) ] (both from # Sigma). These compounds function as dimeric and trimeric substrates, respectively, with the 4-methylumbelliferyl group linked by β-1,4 linkage to the N-acetyl glucosamine (GlcNAc) oligosaccharides. Only 4-methylumbelliferone, when hydrolysed from the GlcNAc oligosaccharides, fluoresces. Simultaneous detection of the various enzyme activities on gels was performed using the method described by Haran et al. (1996) : an overlay gel solution of 1 % agarose (low gel temperature, Sigma) containing 100 m sodium acetate at pH 4±8 was mixed with both enzyme substrates [300 µg ml−" 4-MU-(GlcNAc) and 300 µg ml−" 4-MU-(GlcNAc) ]. After # the gels were overlaid, they were incubated at 37° until bands

Hedva Schickler and others

375

Strain TM pH

4·1

4·2

4·5

4·8

5·1

5·5

6·0

6·3

6·7

M (kDa) 112 84

CHIT102 CHIT73

53

CHIT52 CHIT42

35 29

CHIT33

Strain Y pH

2·1

2·5

3·4

3·9

4·2

4·4

4·7

5·0

5·3

5·7

6·0

6·6

M (kDa) 112 84

CHIT102

CHIT42

53

CHIT42 35

CHIT33

29

Strain T-35 pH

3·1

4·1

4·3

4·6

4·9

5·4

5·9

6·3

6·7

7·2

7·7

8·1

8·4

8·7

M (kDa) 112 84

CHIT102

53

CHIT42

35 29 20

CHIT33

Fig. 2. Two-dimensional gel analysis of T. harzianum chitinolytic profiles. Crude extracellular proteins of T. harzianum grown with chitin as the sole carbon source were separated according to their pI. The pH of each fraction exhibiting chitinolytic activity was measured, and proteins were subjected to SDS–PAGE for second-dimension analysis. Chitinolytic activities were detected after renaturation with a mixture (1 : 1) of the chitin analogs : 4-methylumbelliferyl-N-acetyl-β--glucosaminide (4-MU-GlcNAc) and 4-methylumbelliferyl-β-N,N«-diacetylchitobioside [4-MU-(GlcNAc) ]. Proteins were identified according to their molecular mass (as in Fig. 1). Representative gels # from three runs are shown.

were evident under uv light (302 nm). This experimental system enabled us to follow several chitinolytic activities semi-quantitatively. To estimate the molecular mass of the T. harzianum enzymes accurately, proteins were separated by SDS–PAGE as previously described, on larger gels (18¬11 cm). Molecular masses of the chitinolytic enzymes were estimated from the relationship : log molecular mass of protein standards (lowmolecular protein standards, Bio-Rad) v. distance migrated. Chitinases were designated according to Haran et al. (1995) as CHIT102 and CHIT73 (both N-acetyl-β-glucosaminidases,

EC 3\2\1\30), and CHIT52, CHIT42, CHIT33 and CHIT31 (endochitinases, EC 3\2\1\14). RESULTS AND DISCUSSION Following one-dimensional SDS–PAGE analysis, the chitinolytic system of T. harzianum was found to be composed of 3–6 chitinase activities. However, the differences in the profiles were not sufficient to draw a distinction among the three strains (Fig. 1). To enable the identification of specific strains, we used two-dimensional electrophoresis for protein

Trichoderma identification by chitinase profiles Table 1. pI values of chitinases from strains of T. harzianum Strain Enzyme

TM

Y

T-35

CHIT102 CHIT73 CHIT52 CHIT42 CHIT33

4±8 4±0 4±0 4±0 4±0

4±5 — 4±1 2±5 3±9

6±0 — — 3±1 4±2

analysis. The resultant chitinase profiles of the three Trichoderma strains tested are shown in Fig. 2. As can be clearly seen, the two-dimensional analysis revealed a unique profile for each strain. The pI values of the different chitinases, summarized in Table 1, were calculated as the mean of the pH range in which activity was detected. CHIT73 activity was detected only in strain TM. Its pI value differed from that reported by Lorito et al. (1994) for CHIT73 in T. harzianum strain P1 (pI values of 4±0 and 4±6, respectively). CHIT52 was detected in strains TM and Y as a weak band. This result concurs with the finding that CHIT52 is highly susceptible to both heating and protease activities (Haran et al., 1995). The pI value of CHIT52 was 4±0 in strain TM and exhibited an expanded pH range of 3±4–4±7 in strain Y. The endochitinase CHIT42 has been studied and characterized by several authors. De La Cruz et al. (1992) found its pI value to be 6±2 in strain CECT2413. Harman et al. (1993) found a more acidic pI in strain P1 (pI ¯ 3±9), similar to that found for strain TM (pI ¯ 4±0). Strains Y and T-35 exhibited even more acidic pI values, 2±5 and 3±1, respectively. The only enzyme which exhibited a similar pI value in all strains examined was CHIT33. De La Cruz et al. (1992) also reported the rather close pI value of 4±6 for CHIT33 in strain CECT2413. Following the two-dimensional procedure, CHIT31 activity was not detected in any of the strains examined, and CHIT73 appeared only in strain TM, probably because their renaturation was not complete. Once antibodies against all components of the chitinolytic system of Trichoderma become available, immunodetection of the chitinases after the second dimension may be preferred. Nevertheless, the detected chitinase activities were sufficient for strain identification. Potential biological control agents include rhizospherecompatible fungi and bacteria that exhibit antagonistic activity towards plant pathogens. Before biocontrol can become an important component of plant-disease management, it must be effective, reliable, and economical. Strains within T. harzianum exhibit considerable variability with respect to their biocontrol activity and host range (Sivan & Chet, 1992). For example, one strain of T. harzianum efficiently reduced disease incidence in bean seedlings caused by Rhizoctonia solani, but failed to protect these seedlings from disease caused by Sclerotium rolfsii (Elad et al., 1982). A major stumbling block for the commercial use of Trichoderma is, therefore, the inability to identify the strain needed in a particular application. Moreover, it is important to be able to distinguish the strains which have become established in the field after Trichoderma application. The traditional method of Trichoderma classification by

376 morphological differences (Rifai, 1969) may be useful for laboratory needs, but the manufacture of an industrial product requires a simple and repetitive method, directly related to the biocontrol activity of the fungus. Previous works (Burdon & Marshall, 1983 ; Zamir & Chet, 1985) have made use of enzyme variations in electrophoretically detectable proteins to identify strains. This method is complicated, however, because different enzymes need to be detected with different substrates under different growth conditions. The twodimensional gel analysis of chitinolytic profiles described here overcomes this problem since it involves uniform growth conditions and is easily repeatable. The results described here suggest that in different strains of T. harzianum, chitinolytic enzymes which are of the same mode of action and the same molecular mass exhibit different pI values. pI values of T. harzianum chitinases reported in other strains support this observation. Hence, the twodimensional analysis described is a potentially valuable tool for the identification of specific strains of T. harzianum. This work was supported by a grant from the German–Israeli Foundation for Scientific Research and Development (GIF) and by the Eshkol Fund of the Ministry of Science and Technology. REFERENCES Bradford, M. M. (1976). A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dyebinding. Analytical Biochemistry 72, 248–254. Burdon, J. J. & Marshall, D. R. (1983). The use of isoenzymes in plant disease research. In Isozymes in Plant Genetics and Breeding (ed. S. D. Tanksley & T. J. Orton), 1A, pp. 401–412. Elsevier : Amsterdam. Chet, I. (1987). Trichoderma : application, mode of action, and potential as a biocontrol agent of soilborne plant pathogenic fungi. In Innovative Approaches to Plant Disease Control (ed. I. Chet), pp. 137–160. John Wiley & Sons : New York. Chet, I., Barak, Z. & Oppenheim, A. (1993). Genetic engineering of microorganisms for improved biocontrol activity. In Biological Perspectives in Plant Pathogen Control (ed. I. Chet), pp. 211–235. John Wiley & Sons : New York. De La Cruz, J., Hydalgo-Gallego, A., Lora, J. M., Benitez, T., Pintor-Toro, J. A. & Llobell, A. (1992). Isolation and characterization of three chitinases from Trichoderma harzianum. European Journal of Biochemistry 206, 859–867. Elad, Y., Chet, I. & Henis, Y. (1982). Degradation of plant pathogenic fungi by Trichoderma harzianum. Canadian Journal of Microbiology 28, 719–725. Haran, S., Schickler, H., Oppenheim, A. & Chet, I. (1995). New components of the chitinolytic system of Trichoderma harzianum. Mycological Research 99, 441–446. Haran, S., Schickler, H., Oppenheim, A. & Chet, I. (1996). Differential expression of Trichoderma harzianum chitinases during mycoparasitism. Phytopathology 86, 980–985. Harman, G. E., Hayes, C. K., Lorito, M., Broadway, R. M., Di Pietro, A., Peterbauer, C. & Tronsmo, A. (1993). Chitinolytic enzymes of Trichoderma harzianum : purification of chitobiosidase and endochitinase. Phytopathology 83, 313–318. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lorito, M., Hayes, C. K., Di Pietro, A., Woo, S. L. & Harman, G. E. (1994). Purification, characterization and synergistic activity of a glucan, 1,3-βglucosidase and an N-acetyl-β-glucosaminidase from Trichoderma harzianum. Phytopathology 84, 398–405. McGrew, B. R. & Green, M. (1990). Enhanced removal of detergent and recovery of enzymatic activity following sodium dodecyl sulfate–polyacrylamide gel electrophoresis : use of casein in gel wash buffer. Analytical Biochemistry 189, 68–74. Okon, Y., Chet, I. & Henis, Y. (1973). Effects of lactose, ethanol and

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