In vitro antagonism of cotton seedlings fungi and characterization of chitinase isozyme activities in Trichoderma harzianum

Abstract

The antagonistic fungus Trichoderma harzianum is widely recognized as a potential biocontrol agent against several soil-borne plant pathogens. T. harzinum is rich source of chitinoltic enzymes. In vitro screening of 5 isolates of T. harzinum, one isolate of Chaetomium globosum and one isolate of Conetherium mentance, revealed that all of them had reduced growth area of Macrophomina phaseolina, Fusarium solaniand Rhizoctonia solani on PDA medium, significantly. The inhibition percentage ranged from 77.9 % to 55.9% for M. phaseolina and 59.2% to 40.4% for R. solani by T. harzinum and C. mentance, respectively. Inhibition for F. solani ranged from 76.5% to 55.7% by T. harzinum and C. globosum, respectively. Isozyme gel electrophoresis was used to assess chitinase activity secreted by selected isolates of T. harzinum under different pH degrees and temperatures. Obtained results indicated that activity of chitinase isozyme produced at 30 °C was higher than 15–20 °C for all tested isolates and activity of chitinase produced by isolates No. 4 and 5 of T. harzinum at pH (7–7.5) was higher than at pH 6, respectively.

1. Introduction

Different mechanisms have been suggested as being responsible for their biocontrol activity, which include competition for space and nutrients, secretion of chitinolytic enzymes, mycoparasitism and production of inhibitory compounds (Haram et al., 1996; Zimand et al., 1996).

The chitinolytic system of T. harzianum consists of five to seven distinct enzymes (Haran et al., 1995). 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 (Lorito et al., 1993). The most interesting individual enzyme of the complex is the 42-kDa endochitinase (Ech42).

Ridout et al. (1986) found that 1, 3-beta-d-Glucanase and chitinase activities were induced in T. harizanum when glucose or cells walls of R. solani (anastomosis group AG2) were used as sole C source. Electrophoresis showed that more protein was induced by cell walls than by glucose. The composition, as revealed by electrophoresis of the T. harizanum extra cellular protein was similar when grown on R. solani AG2 and AG4 cell walls, but different for R. solani AG1 cell walls. Several major protein patterns were induced in all strains of T. harizanum but there were strains. Differences in the number and intensity of other protein. The number of induced proteins was less for 4 strain of T. viride and their composition was different from those of 5 strains of T. harizanum, the results indicate that chitinase, 1, 3-beta-d-glucanase and many other extra cellular proteins may be involved in the degradation of R. solani cell walls.

Dipietro et al. (1993) used SDS polyacrylamide gel electrophoresis to assess chitinase activity for Gliocladium virens, the enzyme was active a citric acid and K₃PO₄ buffer over pH 3.5–7.0. in the pH 4.0–6.0 range, the endochitinase showed 90–100% activity, with a maximum at pH 4.5 the optimum temperature for endochitinase activity at pH 4.5 was 30–37 °C, and the activity dropped sharply at temperature above 40 °C possibly due to heat inactivation of the enzyme. The specific activity of the endochitinase at 30 °C was 1 unit per microgram under the assay condition used.

A series of abiotic and biotic environmental parameters has an influence on the biocontrol efficacy of Trichoderma. Some important parameters to be considered are the effects of temperature, water potential and pH (Kredics et al., 2003). A deepest knowledge of the factors that could affect biocontrol activity of Trichoderma would allow us to improve biocontrol conditions for trials at the field level, and to provide information of how the environmental conditions could benefit or decrease the biocontrol effect of T. harzianum (Asran-Amal, 2007). The main aims for the current research, to assess the antagonistic effect of T. harizanum against cotton seedling fungi (2) to molecular characterization of chitinase esozyme activites.

2. Materials and methods

2.1. Fungal isolates

Pathogenic and antagonistic isolates used in this study are listed in Table 1. Isolates of soil-borne fungi were isolated from roots of cotton seedling infected with damping-off disease and collected from cotton-growing areas. Isolation of Trichoderma spp. and other antagonistic fungi were made on potato-dextrose agar (PDA) from healthy cotton rhizosphere grown at agro-climatically different locations. Monosporic cultures were made and stored on PDA slants for further use (Asran-Amal et al., 2005).

Table 1.

Fungal isolates used in the current study.

Fungal isolates	Location	Host
T. harzinum1	Qualybia	Cotton
T. harzinum2	Minufyia	Cotton
T. harzinum3	Faiyum	Cotton
T. harzinum4	Beni-Suyef	Cotton
T. harzinum5	Qualybia	Cotton
Contherium mentance	Unknown	Cotton
Chaetomium globosum	Unknown	Cotton
R. solani	Unknown	Cotton
F. solani	Unknown	Cotton
M. phaseolina	Unknown	Cotton

2.2. In vitro antagonism test

Five isolates of Trichoderma harzinum, one isolate of Contherium mentance and one isolate of Chaetomium globousm isolated from cotton (Gossypium barbardense L.) roots were evaluated in vitro as antagonists against isolates of R. solani, Fusarium solani, and Macrophomina phaseolina. Dual cultures were carried out by using one-week-old cultures of R. solani and T. harzinum on PDA. Agar medium was inoculated with a 5-mm-diameter disc of antagonist positioned diametrically opposite a 5-mm-diameter disc of the pathogen. The distance between discs was approximately 5 cm. Cultures were grown at 28 ± 3 °C, and measurements were taken after four days. In the control treatment, a sterile agar disc (0.5 mm diam.) was placed in dish instead of Trichoderma isolates. There were three replicates for each treatment. At the end of the incubation period, redial growth was measured. The efficiency of Trichoderma in suppressing radial growth was calculated as follows:

(C − T)/C × 100 where C is radial growth measurement of the pathogen in the control and T is radial growth of the pathogen in the presence of Trichoderma spp.

2.3. Chitinase activity as determined by electrophoresis

Fungal strains of T. harzianum were originally isolated from cotton rhizosphere, these strains showed good biocontrol ability against cotton diseases complex. Conidia of the fungus used to inoculated potato-dextrose agar (PDA) plates. For enzyme production, the fungus was grown in 250-ml Erlenmeyer flasks contained 100 ml of a medium which consisted of 680 mg of KH₂PO₄, 870 mg of K₂HPO₄, 200 mg of KCL, 1 g of NH₄NO₃, 200 mg of CaCL₂, 200 mg of MgSO₄·7H₂0, 2 mg of ZnSO₄, 2 mg of MnSO₄, 42 g of moist purified chitin and 5 g of sucrose in 1 L of distilled water final pH 6. The flasks were inoculated with desk from freshly grown PDA plates; the medium was incubated at 15, 20, 25, and 30 °C for 7 days. Anther treatment pH was 6. 5, 7, and 7.5. And the culture filtrate was harvested. (Harman et al., 1993).

One-hundred milligrams of freeze-dried mycelium were homogenized by grinding in liquid nitrogen with a mortar and pestle, and suspended in 1.5 ml of extraction buffer (10 mM Tris–HCl, 1 mM EDTA, 0.1 mM ascorbic acid, pH 7). Extracts were chilled in an ice bath for 1–3 h and centrifuged at 18,000 rpm for 30 min at 4 °C. The supernatant was collected, divided into 100 ml aliquots, stored at −80 °C, and used within 6 months (Aly et al., 2003).

Chitinase staining solution consists of: (10 mM Tris–HCL buffer, pH 7.5, 32 mM N Acytelglucoseamine, 0.18 mg/ml Phosphtedyleserine and 1 mM UDP-N-Acetyle-d-glucoseamine. Staining procedure was done as a fellow: Electrophorized gel was incubated in staining solution at 30 °C for 12. The zone of chitinase activity develops as opaque bands visible in daylight on clean polyacrylamide gel electrophoresis (PAG) background (Kang et al., 1999).

2.4. Gel documentation scanning

Chitinase isozyme gel was scanned densitometrically. The scanning included all major and minor bands in each isozyme. From R_f values which obtained by densitometer scanning for each isozyme, the lowest and the highest R_f values were determined (from 0.01 to 0.99) and presence of a band at a particular R_f was designated as (+), while its absence at the same R_f was designated as (−).

3. Results

3.1. In vitro screening of T. harzinum, C.globosum and C. mentance for antagonism against cotton seedling disease complex

Five isolates of T. harzinum, one isolate of C. globosum and one isolate of C. mentance were in vitro screened for antagonisms against three fungi (M. phaseolina, F. solani and R. solani) involved in damping-off and root rot of cotton seedlings. The antagonists were isolated from cotton rhizosphere. All the antagonistic fungi significantly reduced the growth area of M. phaseolina, F. solani and R. solani on PDA medium. The inhibition percentage for M. phaseolina ranged from 77.9% to 55.9% for T. harzinum and C. mentance, respectively.

The inhibition percentage for F. solani ranged from 76.5% to 55.7% by T. harzinum and C. globosum, respectively. The inhibition percentage for R. solani ranged from 59.2% to 40.4% by T. harzinum and C. mentance respectively. The antagonistic efficiency for fungal antagonist showed that lowest inhibition percentage in R. solani (Table 2) and (Fig. 1). Fig. 2 shows the inhibition of the three pathogen (R. solani A, for F. solani B and M. phaseolina C) in the presence of T. harzinum.

Table 2.

In vitro antagonisms of fungal antagonist against three fungi M. phaseolina, F. solani, and R. solani isolated from cotton.

Fungal antagonist	Geographic origin	Fungal isolates
		M. phaseolina		F. solani		R. solani
		Growth area (cm2)	Inhibition (%)	Growth area (cm2)	Inhibition (%)	Growth area (cm2)	Inhibition (%)
T. harzinum1	Qualybia	31.7	71.6	32.0	71.4	47.3	57.7
T. harzinum2	Minufya	31.0	72.3	27.7	75.2	47.0	58.0
T. harzinum3	Faiyum	38.7	65.4	31.7	71.6	54.3	51.5
T. harzinum4	Beni-Suyef	24.7	77.9	31.3	72.0	46.0	58.9
T. harzinum5	Qualybia	31.3	72.0	26.3	76.5	45.6	59.2
Contherium mentance	Unknown	49.3	55.9	26.3	76.0	66.7	40.4
Chaetomium globosum	Unknown	44.6	60.1	49.6	55.7	61.6	45.0
Control		112	0.0	112	0.0	112	0.0

LSD = 6.266 at (P < 0.05).

Figure 1.

In vitro antagonisms of fungal antagonist against three fungal M. phaseolina, F. solani and R. solani isolates collected form cotton rhizosphere.

Figure 2.

In vitro antagonisms of selected T. harzinum isolate (5) against, A = R. solani, B = F. solani, and C = M. phaseolina.

3.2. In vitro enzymes assay

3.2.1. Chitinase isozymes patterns obtained by polyacrylamide gel electrophoresis (PAGE)

Electrophoretic banding patterns are shows in Figs. 3 and 4 show the phenogram which constructed from cluster analysis of electrophoretic banding patterns of chitinase isozymes in Fig. 3. The tested isolates formed two clusters with similarity level (SL = 29.17), the first cluster (SL = 71.50) consisted of two subclusters. The first one (SL = 92.92) included two isolates from Qualyubiya (isolate T1 and T2), the second sub cluster consisted of two isolate from Beni-Suef, and Faiyoum (isolate T3 and T4) and one isolate from Minufiya (isolate no. T5). The second cluster included only one isolate from C. globosum. The highest (SL = 97.74) was observed between two isolate from Beni-Suyef, and Faiyoum (isolate nos. T3 and T4).

Figure 3.

Chitinase patterns of four isolates Trichoderma harzinum and one isolate Chatomum spp. obtained by polyacrylamide gel electrophoresis (PAGE).

Figure 4.

Dendrogram of chitinase isozyme obtained with polyacrylamide gel electrophoresis (PAGE) for four isolates Trichoderma harzinum and one isolate Chaetomium spp.

3.2.2. Effect of different temperature degrees and pH values on chitinase isozyme activity for the tested isolates of T. harizinum

Data presented in Fig. 5 and Table 3 show the activity of chitinase isozyme produced at different pH and different temperature degrees obtained with polyacrylamide gel electrophoresis (PAGE) for two isolates of T. harzinum (isolates 4 and 5). One parameter was used, i.e. quantity of enzyme/band. T. harzinum (isolates 4 and 5) was grown at pH 6–7.5 (standard pH) gave from 6 to 7 isozymes, while T. harzinum (isolates 4 and 5) was grow at different pH and different temperature gave from 3 to 5 isozymes. The chitinase activity produced at pH 7 were higher than at pH 6 and 7.5 in T. harzinum (isolate no. 5), chitinase activity = 57.63 at R_f 9.4, while the chitinase activity produced at pH 7.5 was higher than at pH 6 and 7 in T. harzinum harzinum (isolate 4), chitinase activity was 57.32 at R_f 10.0. The chitinase activity produced at 30 °C were higher than at 10 °C and 15 °C chitinase activity was 89.47 and 40.49 at R_f 10.2 and R_f 10.0 for T. harzinum (isolates 4 and 5) respectively.

Figure 5.

Effect of different temperatures and pH on isozyme activity on two isolates of Trichoderma harizinum.

Table 3.

Effect of different temperature degrees and pH levels on isozyme activity on two isolates of Trichoderma harizinum.

Rf	Control		Temperature						pH
			T4			T5			T4			T5
			10 °C	15 °C	30 °C	10 °C	15 °C	30 °C	6	7	7.5	6	7	7.5
	Ct	CpH
0.2	2.61	2.17	0.0	0.0	10.53	24.75	39.05	32.04	28.65	32.86	38.59	17.37	10.60	6.18
0.3	0.0	0.0	28.34	2.60	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
0.7	0.0	0.0	0.0	5.65	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
2.1	1.14	4.37	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
5.9	0.0	0.0	15.93	32.14	0.0	0.0	9.82	0.0	0.0	13.50	0.0	0.0	0.0	31.63
6.2	21.89	27.80	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
6.6	0.0	0.0	0.0	0.0	0.0	0.0	6.98	0.0	0.0	0.0	0.0	0.0	0.0	0.0
8.3	0.0	2.27	0.0	0.0	0.0	20.66	0.0	32.58	0.0	0.0	0.0	36.09	0.0	18.87
8.5	4.68	2.08	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
9.4	0.0	0.0	28.62	0.0	0.0	0.0	21.63	0.0	0.0	30.62	0.0	0.0	57.63	36.73
10.00	0.0	0.0	0.0	53.10	0.0	0.0	9.34	40.49	0.0	12.55	57.32	39.67	25.28	0.0
10.02	53.60	48.60	27.11	0.0	89.47	24.67	13.20	0.0	65.53	10.47	0.0	0.0	0.0	0.0
10.25	4.68	11.44	0.0	6.51	0.0	5.35	0.0	3.89	5.82	0.0	4.09	6.87	6.50	6.59

5. Discussion

T. harzinum is a rich source of chitinase enzymes, for control of plant pathogenic fungi. Knowledge concerning the behavior of these fungi as antagonists is essential for their effective use since they can act against target organisms in several ways Jeffries and Young (1994). Most of the Trichoderma strains are mesophilic. Low temperatures in winter may cause a problem during biological control by influencing the activity of the biocontrol agents. Another problem emerging during the application of Trichoderma strains as biocontrol agents is that they cannot tolerate dry conditions. The pH characteristics of the soil also belong to the most important environmental parameters affecting the activities of mycoparasitic Trichoderma strains.

In the present investigation in vitro assay was used to evaluate the antagonism of five isolates of T. harzinum, one isolate of C. globosum, one isolate of C. mentance. Numerous reports indicated that Trichoderma spp. (Ehteshamul et al., 1990; Ehteshamul and Ghaffar, 1992) is effective biocontrol agents. The in vitro test showed that the entire fungal antagonist significantly reduced the colony area of M. phaseolina, F. solani and R. solani. The optimum range for chitinase activity secreted by T. harzinum is pH 6–7, which is comparable to this of other fungi, For example the optimum range of enzyme activity secreted by G. virens ranged from pH 4–6 (Harman et al., 1993).

Isozyme gel electrophoresis was used to assess chitinase activity secreted by selected isolates of T. harzinum under effect different pH and different temperature. Obtained results indicated that, the activity of chitinase produced by T. harzinum isolates Nos. 4 and 5 at pH (7–7.5) was higher than at pH 6, respectively, and the activity of chitinase isozyme produced at 30 °C was higher than but produced at 15–20 °C. The optimum temperature for growth differs among the Trichoderma species. Most Trichoderma strains are mesophilic, and cannot protect germinating seeds from soilborne diseases caused by cold-tolerant strains of plant pathogenic fungi during cold autumn and spring conditions (Kredics et al., 2003). These results are in agreement with for enzyme Dipietro et al. (1993) who showed that the optimum temperature activity at pH 5.5 was 30–37 °C and the activity dropped sharply at temperature above 40 °C. These results of the case effect of different pH on chitinase activity did not agree with these obtained by Harman et al. (1993).