MATERIALS AND METHODS

Fungal isolates: Botrytis cinerea isolates were obtained from decayed tomato (Lycopersicon esculentum) in North-western Algeria. The leaf fragments were placed on filter paper moistened with sterile water in a Petri dish. B. cinerea was cultured on Potato Dextrose Agar (PDA) incubated at 25°C.

Antagonist: The isolate of Trichoderma harzianum, obtained from the laboratory of plant protection (University of Mostaganem, Algeria) was grown in PDA. The media were added with the same concentration of NaCl (50, 100, 150, 300 mEq).

Colony growth inhibition assay with Trichoderma harzianum in dual culture method: Interactions between antagonistic of T. harzianum against Botrytis cinerea were determined by the method described by Dennis and Webster (1971). A 4 mm diameter mycelial disc from the margin of the Trichoderma one week old culture and the pathogen Botrytis cinerea were placed on opposite of the plate at equal distance from the periphery and in control plates only Botrytis cinerea was placed. The plates were incubated at 25±1°C, observed after 4 days and the colony interactions were measured as percentage of inhibition of radial growth of Botrytis cinerea by following equation:

where, L is the percentage inhibition of radial mycelial growth, D1 is radial growth of the pathogen in the control, D2 is radial growth of the pathogen in the presence of T. viride (Edgington et al., 1971).

Evaluation of volatile metabolites: The effect of the volatile metabolites released by T. harzianum on the mycelial growth of the pathogens was evaluated by the inverted plate technique as described by Dennis and Webster (1971). The 5 mm mycelial discs of T. harzianum obtained from the margin of young cultures were placed centrally on the PDA glass dish and incubated in 25±1°C for 72 h. In the control plates, sterile PDA media discs 5 mm in diameter were placed on the plates as mentioned above. At the end of the incubation period, the top of each petri dish was replaced with the bottom of the petri dish inoculated with pathogen and sealed together with adhesive tape. A completely randomized experimental design was used with three replicates. Radial growth of the pathogens was recorded on the 5th day of incubation and L was calculated, as described above.

The influence of NaCl and CaCl₂ on the diameter growth was determined by growing the isolates in a PDA medium at 4 NaCl and CaCl₂ levels (50, 100, 150, 300 mEq), control medium was not amended with salts.

Statistical analysis: All statistical analyses were analyzed by the software of statistics (STATBOX 6.0.4 grimmersoft). The data was analyzed by two-way factorial. Comparison of means and interactions was performed by Duncan’s multiple range tests. Statistical significance was assessed at the level of p = 0.05 or p = 0.01.

RESULTS

Effect of non-volatile metabolites from T. harzianum on the radial growth of B. cinerea: In the absence of salt (Table 1), isolates of B. cinerea do not present the same profile of growth inhibition, the optimum percent for growth inhibition of this fungus was from 63.76-68.15 of F27 and TR46, respectively. In the dual culture experiment, T. harzianum had a marked significant inhibitory effect on the growth of isolates (Table 1). Growth inhibition decreased as follows: TR46 (68.15%)>B27 (66.8 %)>F27 (63.76%). Data in Table 1 indicate that, application of sodium salt caused a significant increase the growth inhibition at various concentrations tested compared with control except TR46 (p>0.05), growth inhibition was decreased for sodium concentrations of 50 and 100 ppm can be obtained from this isolate relative to the control.

Calcium chloride stimulates mycelial growth of T. harzianum. These observations indicate that the growth inhibition of the F27 and B27 isolates might increase (85.01, 70.17%), even at high salinity (150 ppm). However, higher concentrations of calcium chloride (300 ppm) caused F27 and B27 to reduce the percentage of inhibition growth by 81.63 and 65.84%, respectively. Calcium chloride was reducing growth inhibition of the TR46 in the dual culture for all concentrations. The interaction between salt and concentration was significant (p<0.001).

Effect of volatile metabolites from T. harzianum on the radial growth of B. cinere: In the absence of salt, TR46 and F27 was most resistant and revealed lowest percent inhibition of mycelial growth as 52,23% and 53,13, respectively in combination with T. harzianum.

Table 1:	Growth inhibition of three isolates by Trichoderma harzianum in dual culture after incubation at 25°C for 3 days
*,**,***Significant effects at 0.05, 0.01 and 0.001, respectively, NS: Not significant

Table 2:	In vitro growth inhibition of three isolates of Botrytis cinerea by volatile compounds of T. harzianum in PDA medium after incubation at 25oC for 3 days
****,**,***Significant effects at 0.05, 0.01, 0.001, respectively, NS: Not significant

After 3 days of incubation, it is observed that volatile compounds from Trichoderma harzianum exhibited maximum growth inhibition by adding 300 ppm of NaCl to the culture medium when compare to the others salts (Fig. 1). The highest growth inhibition, 94,70, 78,6 and 73,91% was obtained in the high concentrations from isolates B27, TR46 and F27, respectively. The interaction between salt and concentration was significant for all salts isolates (p<0.05) (Table 2). In the presence of low salt concentrations (50 and 100 ppm), the percentage of inhibition growth due to the interaction between fungi and T. harzianum was still very important with CaCl₂ more than NaCl. However, the antifungal activity of volatile metabolites produced by T. harzianum showed 90.037% maximum inhibition of mycelia growth with B27 in presence of higher concentrations of calcium chloride (300 ppm). On the other hand the effect of CaCl₂ on T. harzianum volatile metabolites action on the growth of B27 isolat presented in Table 2 showed that the volatile metabolic substances of antagonistic present the same profile of the percentage inhibition of mycelia growth in the different concentrations tested.

Fig. 1:	Antagonistic effect of the isolates on Botrytis cinerea in dual culture assay at 25°C on potato dextrose agar media

DISCUSSION

In the present study, we tested effect of salinity on in vitro Trichoderma harzianum antagonism against B. cinerea for their production of volatile and non-volatile compounds.

In the absence of salt, The direct confrontation of T. harzianum against the different isolates of B. cinerea in vitro on PDA medium showed that T. harzianum inhibited the growth of pathogenic fungi at varying degrees.

The non-volatile secondary metabolites from T. harzianum were found more effective in suppressing the mycelial growth of all isolate when compared to volatile compounds except B27 isolate, growth inhibition was recorded by volatile compounds up to 79,81%.

The inhibition in radial growth of two interacting organisms in dual culture has been attributed to secretion of extracellular hydrolytic enzymes (Schirmbock et al., 1994) by the production of antibiotics Howell (1998) or as well as some cell walls degrading enzymes such as chitinases, glucanases that break down polysaccharides, chitins and þ-glucanase, there- by destroying cell wall integrity (Elad, 2000). Trichoderma species are known to produce a number of antibiotics such as Trichodernin, Trichodermol, Harzianum A and Harzianolide (Dennis and Webster, 1971; Kucuk and Kivanc, 2004).

Ajith and Lakshmidevi (2010) examined the potential of Trichoderma harzianum which suppress the mycelia growth of Colletotrichum capsici causing anthracnose on bell peppers. Doi and Mori (1994) reported volatile compounds produced from Trichoderma species were able to arrest and inhibit the hyphal growth of various plant pathogenic fungi. Amin et al. (2010) reported the effectiveness of volatiles produced by T. harzianum (Th-1) causing 53.63% inhibition of Colletotrichum capsici.

In the case of the evaluation the effect of NaCl and CaCl₂ on the Trichoderma harzianum against B. cinerea, salinity is most favorable to the increase growth of Trichoderma harzianum, especially at high concentrations. T. harzianum was able to tolerate the presence of salt in the medium culture even at high concentrations.

The analysis shows that CaCl₂ stimulate the development of the Trichoderma harzianum as compared to sodium chloride.

Our data indicate that volatile compounds from Trichoderma were found more effective in suppressing the mycelial growth of all fungal pathogens when compared to non-volatile metabolites.

In indirect confrontation, the effect of salinity on T. harzianum volatile compounds action is perceived in salt concentrations demonstrate that T. harzianum was able to colonize the space and to inhibit the mycelial growth of Botrytis cinerea. It was found that NaCl inhibited the growth of pathogen (94,70%) more than CaCl₂ (92,78%).

In the direct confrontation, the growth of Botrytis cinerea was intensely inhibited by non-volatile metabolites was stimulated by salt and moderately inhibited the pathogen growth. The percentage of growth inhibition varied between 65.17 and 82, 12% for NaCl and 61.19-85.01 in the presence of CaCl₂.

The growth and multiplication of T. harzianum were stimulated by the addition of salt to the growth media, it showed also some good antagonist abilities at the high concentrations of salinity demonstrated by confrontation techniques. The strain activity of T. harzianum was able to tolerate the presence of salt in the medium culture even at high concentrations. Benyahyia (1998) showed that increasing the salinity of the medium promotes the in vitro mycelia growth of Phytophthora citrophthora and P. parasitica agents of root rot of citrus with an optimum between -1.44 and -3.11 bars. The mycelium growth of T. harzianum developed in saline media between 2 and 6 g L‾¹ of NaCl doses not differ from the control cultures without salt. Nevertheless, the capacity C declines significantly starting from 6 g L‾¹ of NaCl but C value remain high (80%), showing an important capacity of the antagonist to cover the colony of the pathogen in presence of salt (Regragui and Lahlou, 2005).

Growth inhibition of the pathogens by the Trichoderma metabolites has been reported by several workers., such as Fusarium oxysporum, Rshizoctonia solani, Curvularia lunata, Bipolaris sorokiniana and Colletotrichum lagenarium, Colletotrichum acutatum, Colletotrichum gloeosporioides (Yang et al., 2006; Zivkovic et al., 2010). The antagonistic activity of Trichoderma depends on multiple synergistic mechanisms (Nallathambi et al., 2009; Howell, 2003). The various mechanisms include antibiosis, parasitism, inducing host-plant resistance, competition and secretion of chitinolytic enzymes, mycoparasitism and production of inhibitory compounds (Harman et al., 2004). Also it is found that there is large variety of volatile secondary metabolites produced by Trichoderma such as ethylene, hydrogen cyanide, aldehydes and ketones which play an important role in controlling the plant pathogens (Vey et al., 2001). Previous studies have demonstrated that before mycelia of fungi interact, Trichoderma sp. produces low quantities of extracellular exochitinases (Kullnig et al., 2000; Brunner et al., 2003). The diffusion of these enzymes dissolves cell fragments of host cells. These cell fragments in turn induce the production of further enzymes and trigger a cascade of physiological changes, stimulating rapid and directed growth of Trichoderma sp. (Zeilinger et al., 1999).

Although, Trichoderma sp. species is widely used to control many plant pathogens but not much work has been done to give the effect of salinity on the antagonist effect of Trichoderma sp. Knowledge of soil salinity and its potential effects on the antagonist against diseases is essential for disease management strategies. Therefore, it is important that biocontrol potential of Trichoderma harzianum under salinity conditions should be further evaluated in vivo against Botrytis cinerea causing grey mould disease.