In vitro water activity and pH dependence of mycelial growth and extracellular enzyme activities of Trichoderma strains with biocontrol potential*

Authors


  • *

    In memoriam Prof. Ferenc Kevei (1942–2003).

László Kredics, HAS-USZ Microbiological Research Group, PO Box 533, H-6701 Szeged, Hungary (e-mail: kredics@bio.u-szeged.hu).

Abstract

Aims:  Water activity (aw) and pH are probably the most important environmental parameters affecting the activities of mycoparasitic Trichoderma strains. Therefore it is important to collect information on the effects of these factors on mycelial growth and on the in vitro activities of extracellular enzymes involved in nutrient competition (e.g. β-glucosidase, cellobiohydrolase and β-xylosidase) and mycoparasitism (e.g. N-acetyl-β-glucosaminidase, trypsin-like protease and chymotrypsin-like protease) of Trichoderma strains with biocontrol potential.

Methods and Results:  Water activity and pH dependence of the linear mycelial growth of five examined Trichoderma strains belonging to three different species groups was examined on yeast extract and soil extract media. Maximal growth rates were observed at aw 0·997 and pH 4·0 in the case of all strains. The activities of the examined extracellular enzymes at different aw and pH values were determined spectrophotometrically after incubation with chromogenic p-nitrophenyl and p-nitroaniline substrates. Maximal enzyme activities were measured at aw 0·950 for β-glucosidase, trypsin-like protease and chymotrypsin-like protease, at 0·910 for cellobiohydrolase and at 0·993 for β-xylosidase and N-acetyl-β-glucosaminidase enzymes. Optimal pH values are suggested to be at 5·0 for β-glucosidase, cellobiohydrolase and N-acetyl-β-glucosaminidase, at 3·0 for β-xylosidase, at 6·0 for trypsin-like protease and between 6·0 and 7·0 for chymotrypsin-like protease activities, respectively.

Conclusions:  Extracellular enzymes of the examined mycoparasitic Trichoderma strains are able to display activities under a wider range of aw and pH values than those allowing mycelial growth.

Significance and Impact of the Study:  Data about the effects of aw and pH on mycelial growth and extracellular enzyme activities of Trichoderma reveal useful information about the applicability of biocontrol strains in agricultural soils with specific water and pH relations.

Introduction

Trichoderma species are imperfect fungi, with teleomorphs belonging to the ascomycete order Hypocreales. Some Trichoderma species are very good cellulase producers and therefore they are important for the biotechnological industry (Kubicek et al. 1990; Réczey et al. 1996; Juhász et al. 2003). The agricultural importance of the genus Trichoderma is that some of its members possess mycoparasitic abilities against plant pathogenic fungi, which allows for the development of biocontrol strategies based on Trichoderma strains (Papavizas 1985; Benítez et al. 1998; Manczinger 1999; Manczinger et al. 2002a). Such strategies can be incorporated in a complex integrated plant protection. When planning the application of biocontrol strains, it is very important to consider the environmental stresses affecting microbial activities. The study of the influence of environmental parameters on Trichoderma strains is of great importance: biocontrol strains should have better stress tolerance levels than the plant pathogens against which they are going to be used during biological control. Besides the effects of low temperature (Magan 1988; Eastburn and Butler 1991; Antal et al. 2000), heavy metals (Babich et al. 1982; Somashekar et al. 1983; Frank et al. 1993; Kredics et al. 2001a,b), pesticides (Stratton 1983; Beatty and Sohn 1986) and antagonistic bacteria (Naár and Kecskés 1998; Manczinger et al. 2002b) on biocontrol Trichoderma strains, it is also important to consider the effects of water relations and pH on mycelial growth and extracellular enzyme systems.

In addition to the effect of cold winters in temperate climates, water conditions are among the crucial factors affecting microbial activity in natural soil systems. Dry conditions may occur even in usually damp soils as a result of normal drying between rain periods. Moreover, there are phytopathogens with the ability to grow and cause disease even in dry soils. Therefore it is reasonable to study the influence of water conditions on biocontrol Trichoderma strains before their practical application. Data are available about the influence of water availability on the enzyme biosynthesis and enzyme activities produced by T. viride in solid-state fermentation (Grajek and Gervais 1987), on spore germination and germ tube growth of Trichoderma species (Magan 1988), on growth, competitive and saprophytic abilities of T. harzianum (Badham 1991; Eastburn and Butler 1991), and about the in vitro effects of water relations on growth, enzyme secretion and extracellular enzyme activities of a T. harzianum strain with biocontrol potential (Kredics et al. 2000). However, there is a lack of comparative information about the effects of water activity (aw) on mycoparasitic Trichoderma strains belonging to different species groups.

Trichoderma strains with biocontrol potential are applied in agricultural soils with certain pH characteristics. Therefore, it is also of great importance to collect information about the effects of pH on mycelial growth and extracellular enzyme activities of the biocontrol strains.

This study was designed to examine the effects of different aw and pH values on linear mycelial growth and extracellular enzyme activities of cold tolerant, mycoparasitic Trichoderma strains belonging to different species aggregates.

Materials and methods

Strains and culture media

Strains T. aureoviride T122, T. harzianum T66 (ATCC MYA-1175) and T334, T. viride T114 and T228 with biocontrol potential against phytopathogenic Fusarium, Pythium and Rhizoctonia species were isolated from the soil of the forest at Ásotthalom (southern Hungary). All of them were characterized as cold-tolerant strains (Antal et al. 2000). The Trichoderma strains were maintained on minimal agar medium (Manczinger and Ferenczy 1985).

Depending on the enzyme activities to be examined, 1% microcrystalline cellulose, xylan, chitin or skim milk powder (Sigma) was incorporated as inducers in 20 ml liquid media (5 g l−1 KH2PO4, 1 g l−1 NaNO3, 1 g l−1 MgSO4·7H2O in distilled water). In 50 ml Erlenmeyer flasks, these solutions were inoculated with conidial suspensions of the examined Trichoderma strains to a final concentration of 105 conidia per millilitre, and incubated on a shaker at 200 rev min−1 and 25°C. After 4 days of incubation, the mycelial pellets were removed by centrifugation, and enzyme activities were measured in the supernatants.

Control of aw and pH

Using NaCl aw was controlled in agar plates according to the data published earlier (Chirife and Resnik 1984). The use of glycerol as an osmoticum was avoided in the culture media because of its potential to serve as a carbon source, but was used for the investigation of the effect of aw on in vitro enzyme activities at different temperatures in order to reach lower water activities than possible with NaCl. Glycerol concentrations were calculated using the Norrish equation (Norrish 1966) with K = 1·16. Osmolality of the solutions was determined by osmometric measurement using a Vogel OM 801 osmometer (Vogel Medizinische Technik und Elektronik, Giessen, Germany). aw was calculated from the osmolality values as described earlier (Kredics et al. 2000).

McIlvain buffer solutions (mixtures of 0·2 mol l−1 Na2HPO4 and 0·1 mol l−1 citric acid in different proportions) resulting in pH values between 2·0 and 9·0 were applied for the control of pH both in solid media and in the enzymatic reactions.

Growth measurement

For the determination of radial growth rates, Trichoderma strains were inoculated centrally with 4-mm-diameter plugs, cut from the margin of actively growing colonies, onto Petri plates containing yeast extract agar (10 g l−1 glucose, 5 g l−1 KH2PO4, 1 g l−1 NaNO3, 2 g l−1 yeast extract, 1 g l−1 MgSO4·7H2O, 15 g l−1 agar in distilled water) or soil extract medium. For the preparation of soil extract, 1 kg agricultural soil was suspended in 4 l distilled water. After sedimentation, the liquid phase was used to prepare the medium. The media were supplemented with NaCl at different concentrations for the examination of the effects of aw in the range of 0·997–0·922. To determine the pH dependence of mycelial growth, media were buffered to different pH values between 2·0 and 9·0 with McIlvain buffer solutions. During incubation at 25°C, colony radii were measured daily. The examinations were carried out with two replicates for each strain and treatment, the standard deviation values (s.d.) were determined.

Spectrophotometric measurement of extracellular enzyme activities

Total activities of β-glucosidase (EC 3.2.1.21), cellobiohydrolase (EC 3.2.1.91), β-xylosidase (EC 3.2.1.37), N-acetyl-β-glucosaminidase (EC 3.2.1.52), trypsin-like protease (EC 3.4.21.4) and chymotrypsin-like protease (EC 3.4.21.1) were assayed using p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl-β-d-cellobioside, p-nitrophenyl-β-d-xylopiranoside, p-nitrophenyl-N-acetyl-β-d-glucosaminide, N-benzoyl-Phe-Val-Arg-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma) substrates, respectively. For the study of the effects of aw, substrates were dissolved in glycerol–distilled water solutions to obtain water activities between 0·993 and 0·860. McIlvain buffer solutions resulting in pH values between 2·0 and 9·0 were used to dissolve the substrates in the experiments designed for the investigation of the effects of pH on extracellular enzyme activities. The end concentrations of the substrates in the reaction mixtures were 100 μg ml−1. Incubation was performed at 25°C for 1 h. In the case of p-nitrophenyl derivatives, the enzymatic reactions were stopped with 50 μl 100 g l−1 Na2CO3. The O.D. of the samples was determined with a Labsystems Uniskan II microtiter plate spectrophotometer (Labsystems, Helsinki, Finland) at a wavelength of 405 nm. Statistical analysis was performed on the measured values using two factor anova carried out with the software R (Ihaka and Gentleman 1996). The effect of aw (factor 1) on the in vitro extracellular enzyme activities was examined in three replicate series which were considered as blocks (factor 2). Within the blocks there were no replicates. The same statistical method was used for the treatment of the data on the pH dependence of the examined extracellular enzymes.

Results

Effect of aw and pH on mycelial growth of mycoparasitic Trichoderma strains

Radial growth rates of the examined strains were determined at five different aw values on yeast extract and soil extract media (Table 1). The highest aw applied (0·997) seemed to be optimal for the growth of all strains on both types of media. Colony growth decreased with the decrease in aw. Only limited growth was observed at 0·922 in the case of three examined strains on yeast extract agar, while the mycelial growth of all examined strains ceased already at 0·962 on soil extract agar. The examined strains were able to grow on a wide range of pH from 2·0 to 6·0 (Table 2), and the maximal growth was observed under acidic conditions at pH 4·0 on both types of media. The mycelial growth ceased at pH 8·0 and 7·0 on yeast and soil extract agar, respectively.

Table 1.  Effect of aw on linear mycelial growth of mycoparasitic Trichoderma strains on yeast extract and soil extract media. Radial growth rates in mm day−1 ± s.d.
Strainsaw
0·9970·9800·9620·9450·922
  1. Data from yeast extract and soil extract media are set in bold and italic, respectively.

T. aureoviride T12220·13 ± 0·1310·88 ± 0·135·11 ± 0·042·15 ± 0·000·25 ± 0·00
7·20 ± 0·001·13 ± 0·000·00 ± 0·000·00 ± 0·000·00 ± 0·00
T. harzianum T6621·38 ± 0·3811·63 ± 0·135·82 ± 0·042·08 ± 0·030·00 ± 0·00
7·20 ± 0·01·31 ± 0·060·00 ± 0·000·00 ± 0·000·00 ± 0·00
T. harzianum T33418·13 ± 0·1311·00 ± 0·005·22 ± 0·002·13 ± 0·030·00 ± 0·00
6·45 ± 0·051·88 ± 0·060·00 ± 0·000·00 ± 0·000·00 ± 0·00
T. viride T11418·25 ± 0·259·50 ± 0·006·70 ± 0·002·03 ± 0·030·35 ± 0·00
7·80 ± 0·200·88 ± 0·000·00 ± 0·000·00 ± 0·000·00 ± 0·00
T. viride T22820·25 ± 0·0011·13 ± 0·135·50 ± 0·002·23 ± 0·030·33 ± 0·03
6·75 ± 0·051·38 ± 0·000·00 ± 0·000·00 ± 0·000·00 ± 0·00
Table 2.  Effect of pH on linear mycelial growth of mycoparasitic Trichoderma strains on yeast extract- and soil extract media. Radial growth rates in mm day−1 ± s.d.
StrainspH
2·03·04·05·06·07·08·09·0
  1. Data from yeast extract and soil extract media are set in bold and italic, respectively.

T. aureoviride T1229·75 ± 0·2516·25 ± 0·0018·75 ± 0·2515·00 ± 0·0010·00 ± 0·002·50 ± 0·000·00 ± 0·000·00 ± 0·00
4·13 ± 0·046·17 ± 0·007·38 ± 0·136·63 ± 0·042·29 ± 0·130·00 ± 0·000·00 ± 0·000·00 ± 0·00
T. harzianum T669·94 ± 0·0715·00 ± 0·0016·63 ± 0·1313·50 ± 0·178·00 ± 0·340·00 ± 0·000·00 ± 0·000·00 ± 0·00
4·08 ± 0·086·33 ± 0·007·29 ± 0·136·83 ± 0·002·17 ± 0·000·00 ± 0·000·00 ± 0·000·00 ± 0·00
T. harzianum T3349·32 ± 0·0714·09 ± 0·0914·88 ± 0·1314·09 ± 0·099·67 ± 0·000·71 ± 0·130·00 ± 0·000·00 ± 0·00
4·21 ± 0·046·04 ± 0·046·63 ± 0·046·50 ± 0·002·29 ± 0·040·00 ± 0·000·00 ± 0·000·00 ± 0·00
T. viride T1149·50 ± 0·1317·00 ± 0·0019·75 ± 0·0015·00 ± 0·0011·67 ± 0·002·29 ± 0·000·00 ± 0·000·00 ± 0·00
3·88 ± 0·046·25 ± 0·007·21 ± 0·046·50 ± 0·082·88 ± 0·040·00 ± 0·000·00 ± 0·000·00 ± 0·00
T. virideT2289·32 ± 0·0717·50 ± 0·0018·00 ± 0·0016·25 ± 0 0010·34 ± 0·002·29 ± 0·000·00 ± 0·000·00 ± 0·00
4·13 ± 0·045·88 ± 0·047·25 ± 0·006·54 ± 0·042·45 ± 0·040·00 ± 0·000·00 ± 0·000·00 ± 0·00

Water activity dependence of total activities of Trichoderma extracellular enzymes

The effect of aw on the in vitro activities of Trichoderma extracellular enzymes involved in competition (β-glucosidase, cellobiohydrolase and β-xylosidase) and mycoparasitism (N-acetyl-β-glucosaminidase, trypsin-like protease and chymotrypsin-like protease) was studied at four aw values. Relative enzyme activities were calculated from the data of each replicate series: the maximal enzyme activities measured were taken as 100%. Figure 1 shows the means of the relative enzyme activities from the three replicate series at each examined aw value. The in vitro activities of the examined extracellular enzymes proved to be dependent on aw, corresponding P-values of anova are indicated in Fig. 1. In the case of β-xylosidase (Fig. 1c) and N-acetyl-β-glucosaminidase (Fig. 1d) enzymes, the maximal in vitro enzyme activities were measured at 0·993. Cellobiohydrolase activities (Fig. 1b) had their maximum at 0·91, while maximal activities of β-glucosidase (Fig. 1a), trypsin-like protease (Fig. 1e) and chymotrypsin-like protease (Fig. 1f) were detected at 0·95. All of the examined enzymes displayed in vitro activities even at the aw value of 0·860.

Figure 1.

Effect of aw on the in vitro extracellular enzyme activities of mycoparasitic Trichoderma strains. Relative enzyme activities are presented in the percentage of the maximal activities measured. (a) β-glucosidase, (b) cellobiohydrolase, (c) β-xylosidase, (d) N-acetyl-β-glucosaminidase, (e) trypsin-like protease, (f) chymotrypsin-like protease. Corresponding P-values of anova are indicated

pH dependence of total activities of Trichoderma extracellular enzymes

The effect of pH on the in vitro activities of Trichoderma extracellular enzymes was studied at pH values ranging from 2·0 to 9·0. Relative enzyme activities calculated from the measured values are presented in Fig. 2. pH had an effect on the in vitro activities of all examined enzymes, corresponding P-values of anova are indicated in Fig. 2. The pH dependence of β-glucosidase (Fig. 2a), cellobiohydrolase (Fig. 2b) and N-acetyl-β-glucosaminidase (Fig. 2d) activities suggest optimum trends with maxima at pH 5·0. In the case of N-acetyl-β-glucosaminidase, the presence of another peak is suggested at pH 9·0. Maximal β-xylosidase activities (Fig. 2c) were measured under acidic conditions (pH 3·0), but activities were present even at pH values up to 9·0. The maximal trypsin-like (Fig. 2e) and chymotrypsin-like (Fig. 2f) protease activities were detected at pH 6·0 and 6·0–7·0, respectively.

Figure 2.

Effect of pH on the in vitro extracellular enzyme activities of mycoparasitic Trichoderma strains. Relative enzyme activities are presented in the percentage of the maximal activities measured. (a) β-glucosidase, (b) cellobiohydrolase, (c) β-xylosidase, (d) N-acetyl-β-glucosaminidase, (e) trypsin-like protease, (f) chymotrypsin-like protease. Corresponding P-values of anova are indicated

Discussion

Maximal activities of β-glucosidase, cellobiohydrolase and both proteases were detected at lower aw values than those optimal for mycelial growth. In the case of other examined extracellular enzymes, the aw values optimal for mycelial growth and in vitro enzyme activities were similar. However, all of the examined enzymes displayed activities even at aw values where mycelial growth has already ceased. More detailed data are available in the case of T. harzianum T66 about the dependence of mycelial growth, enzyme secretion and in vitro activities of extracellular enzymes on water relations at different temperatures: the secretion of cellobiohydrolase and N-acetyl-β-glucosaminidase enzymes was optimal at the highest examined water potential, while the maximum activities of secreted β-glucosidase, β-xylosidase and chymotrypsin-like protease enzymes occurred at lower water potential values than those optimal for growth. The in vitro enzyme activities were also affected by water potential, but significant enzyme activities were measured for most of the enzymes even at water potential values below the limit of mycelial growth (Kredics et al. 2000). Based on this earlier study and the present results, there is a possibility of using mutants with improved xerotolerance for biocontrol purposes in soils with lower water availabilities.

The examined strains were able to grow in a wide range of pH from 2·0 to 6·0 with maximal growth rates at 4·0. Jackson et al. (1991) found that optimum biomass production of three Trichoderma isolates occurred at pH values between 4·6 and 6·8.

In an earlier study, extracellular enzyme profiles of strains T. aureoviride T122, T. harzianum T66 and T. viride T228 were determined by Sephadex G150 column chromatography (Antal et al. 2001). Both the β-glucosidase and cellobiohydrolase isoenzyme profiles of these three strains showed at least two isoenzymes. Two β-glucosidases were found in T. pseudokoningii by Dong et al. (1997), and two cellobiohydrolase enzymes were described in the case of T. koningii (Wey et al. 1994), T. viride (Wang et al. 1995) and T. reesei, a species of industrial importance with a well characterized cellulolytic enzyme system (Kubicek et al. 1990). Enzyme preparations purified from T. harzianum and T. longibrachiatum were found to display β-glucosidase activities with pH optima at 5·0–7·0 (Kalra and Sandhu 1986) and 5·5 (Kalra et al. 1986), respectively. Chen et al. (1992) purified and characterized two extracellular β-glucosidases from T. reesei with pH optima at 4·6 and 4·0 for β-glucosidase I and II, respectively. The T. reesei cellobiohydrolase II has been found to retain maximal activity in a broad pH range between 2·5 and 6·5 (Teleman et al. 1998). According to the results of this present study, the pH optima of both supposed β-glucosidase and cellobiohydrolase isoenzymes of the examined Trichoderma strains seem to be near pH 5·0.

The β-xylosidase activities of strains T122, T66 and T228 were detected as multiple peaks by Sephadex G150 chromatography (Antal et al. 2001). Our present results also suggest the presence of more isoenzymes with different pH relations. A β-xylosidase stable at pH 2·5–7·4 with an optimum in the range of 3·5–4·0 was purified and characterized from T. koningii by Li et al. (2000). The β-xylosidase of T. reesei is stable between pH 3·0 and 6·0 with an optimum at 4·0 (Herrmann et al. 1997). β-Xylosidase activities optimal between pH 4·0 and 4·5 were reported from T. harzianum by De A. Ximenes et al. (1996).

The two peaks detected in the pH profile of N-acetyl-β-glucosaminidase suggest the possibility for the presence of isoenzymes with different pH dependence in the case of our examined strains. An N-acetyl-β-glucosaminidase with optimal pH at 4·0 was purified from T. reesei (Nogawa et al. 1998). In T. harzianum two N-acetyl-β-glucosaminidases were described earlier (Ulhoa and Peberdy 1991; Lorito et al. 1994; Haran et al. 1995), one of the isoenzymes was characterized with maximal activities at pH 5·5 (Ulhoa and Peberdy 1991).

The Sephadex G150 profiles of trypsin-like and chymotrypsin-like proteases were found to be complex in the case of strains T122, T66 and T228, both systems seemed to be consisting of more isoenzymes (Antal et al. 2001). The high activities of trypsin-like protease measured in the pH range between pH 5·0 and 9·0 suggest the presence of isoenzymes with different pH optima. The optimal pH for the measurement of extracellular Trichoderma proteases were found to be 7·0 by Mischke (1996). Individual serine proteases stable within the pH range of 4·0–11·0 with an optimum at 10·5 have been isolated from T. lignorum and T. koningii (Gajda et al. 1981). Optimal pH values of the proteolytic activity of T. viride in mixed cultures with Sclerotium rolfsii in soil were 5·5–6·5 with a maximum at 6·0 (Rodriguez-Kabana et al. 1978).

Enzyme systems with supposed roles in mycoparasitism and the degradation of host cells (N-acetyl-β-glucosaminidase, trypsin-like protease and chymotrypsin-like protease) were active under a wide range of pH, even at alkalic values, where mycelial growth was already inhibited (pH 8·0–9·0). Similar results were obtained for β-xylosidase, however, the optimal pH range of the cellulolytic β-glucosidase and cellobiohydrolase enzymes seemed to be narrower, suggesting, that pH values between pH 4·0 and 6·0 are optimal for effective nutrient competition.

Adaptation of the extracellular enzyme systems to environments with different aw and pH characteristics seems to be an important mechanism of evolution enabling the effective mycoparasitism and competition for nutrients under a wider range of these environmental parameters. However, the examined extracellular enzymes of mycoparasitic Trichoderma strains remain active even at aw and pH values, which are already inhibiting mycelial growth. Similar results were found in the presence of several metal compounds (Kredics et al. 2001a,b). Therefore the improvement of stress tolerance in Trichoderma strains could result in biocontrol agents effective against plant pathogenic fungi even under environmental conditions which are unfavourable for the wild type strains.

The results presented in this study about the effects of aw and pH on mycelial growth and extracellular enzyme activities of mycoparasitic Trichoderma strains may reveal useful information for prediction of the potential applicability of biocontrol strains in agricultural soils with certain water and pH relations.

Acknowledgements

We thank Miss Mária Lele and Mr Gergely L. Nagy for their technical help. This work was supported by grants F037663 of the Hungarian Scientific Research Fund and grant OMFB-00219/2002 of the Hungarian Ministry of Education.

Ancillary