MATERIALS AND METHODS

Isolation, purification and identification of the pathogen: The diseased leaves of faba bean plants were collected from different sites of Mansoura districts in Dakahlia governorate, Egypt, namely Sinbellawen, Aga, Talkha, Temay El-Amded, Mansoura and Bany Ebeed by sterile water. The leaves were cutted into small pieces (1-2 cm²), disinfected in 0.01% HgCl₂ for 1-3 min for surface sterilization. Surface sterilized pieces were then rinsed in sterilized water several times to remove the remaining disinfectant solution, dried on sterilized filter papers. Using sterilized forceps, the dried pieces were then transferred into petri-dishes containing Potato Dextrose Agar (PDA) medium supplemented with 200 ppm streptomycin sulphate to inhibit bacterial contamination. The dishes were then incubated at 24-26°C for 7-10 days and checked for microbial growth two days after inoculation. Purification of the resulting isolates was done by using single spore technique to obtain them in pure cultures¹².

Pure cultures of the isolated fungus were identified according to the cultural characteristics, morphological and microscopic features (mycelial development and spore formation) described by Ellis¹³. Stock cultures were stored on PDA medium under sterile mineral oil at 4°C.

Pathogenicity test: The different Botrytis fabae isolates were grown on PDA medium and incubated at 20°C for 12 days¹⁴ under light regime of UV and normal fluorescent (12 h/12 h) to enhance sporulation. Conidia were harvested and transferred to 10 mL of sterilized water for each plate. Conidial concentration was estimated using a haemocytometer and adjusted to about 5×10⁴ spores mL^–1.

Giza 40 seeds of faba bean were sown in plastic pots (35 cm in diameter) containing 12-13 kg clay: sand (3:1, v/v) soil. Five seeds were sown in each pot, plants were left to grow and watered when necessary. After 45 days, the intact plants were sprayed with the spore suspension of the pathogen and covered with polyethylene bags to maintain enough humidity around the plants. Control plants were sprayed with sterilized water. All pots were kept in a glass house under natural conditions. Disease assessment was recorded after 10 days of microbial spraying.

The Disease Severity (DS) was recorded according to the disease index which based on the standard scale of Gondran¹⁵ as follows: (1) Healthy plants, (2) Small spots, (3) Increasing spots number and spreading, (4) Coalesce spots together and about ¼ of leaf surface is necrotic, (5) Half of the leaf surface is necrotic and (6) Leaves die and fall.

Antagonistic effect of Trichoderma harzianum against Botrytis fabae )dual culture test): The isolate of Trichoderma harzianum which is used through the present study was obtained from the Department of Biological Control, Institute of Plant Pathology, Giza, Egypt.

The antagonistic effect of Trichoderma harzianum was investigated using dual culture technique¹⁶. Six millimeter diameter discs taken from the growing edge of 5 days old culture of B. fabae were placed 10 mm from the edge of each 90 mm PDA plate. Six millimeter diameter discs taken from the growing edge of 4 days old culture of T. harzianum were also placed 10 mm from the edge of the plate oppositely to B. fabae. A control PDA plates inoculated with 6 mm diameter discs of B. fabae or T. harzianum were used. All plates were incubated at 26±2°C under alternating luminosity (12 h light/12 h darkness) for 8 days. The inward linear growth (distance between the center of the disc and the edge of the colony) was measured after 2, 4, 6 and 8 days and the interaction between the two fungi was recorded either in the form of inhibition zone or overgrowth of T. harzianum on B. fabae¹⁷.

Antifungal activity of non-volatile metabolites of Trichoderma harzianum against B. fabae: The effect of non-volatile metabolites from Trichoderma sp., against B. fabae was tested by the method described by Kucuk and Kivanc¹⁸. Initially, mycelial agar plugs (6 mm diameter) taken from the edge of a young culture of T. harzianum were transferred to the center of petri-dishes (90 mm diameter) containing 20 mL PDA sterilized medium covered by a sterilized cellophane membrane. The plates were incubated at 25°C for 72 h. Then the cellophane membrane covered with Trichoderma mycelial growth was removed before the growth reaches the edges of the plates. On the same media a disc of 6 mm diameter taken from the growing edge of B. fabae culture 5 days old was placed. Then the plates were incubated at 22°C for a further 6 days. Control plates of B. fabae growing on PDA medium were used for comparison. Five replicates (plates) were used for each treatment. The mean diameter of B. fabae was measured and the percentage of growth inhibition due to the non-volatile metabolites from Trichoderma was calculated.

Preparation of specimen for Scanning Electron Microscopy (SEM): The PDA plate was inoculated at a constant distance from the edge of the petri dish with a mycelial disc (5 mm) cut from the leading edge of both colonies of T. harzianum and B. fabae. Both of the two fungi grew toward each other and their hyphae intermingled. After 6 days of incubation, the plate cultures were observed under a light microscope to verify the early stage of interaction. The interaction sites were marked and agar blocks of 1 cm² were removed for SEM preparation. Sample preparation was performed using the tissue processor model Lynexel, Leica. Where the mycelial samples from the interaction region were fixed with osmium oxide and then dehydrated using serial dilution of ethyl alcohol then finally acetone. The processed samples were then dried using a critical point drier (EMS 850), coated with gold using sputter coater (EMS 550) then the samples were examined using a SEM (JEOL100CX-ASID-4D)¹⁹.

RESULTS

Pathogenicity test: The results illustrated in Table 1 indicated that all B. fabae isolates have the potency to cause faba bean chocolate spot disease, where disease incidence ranged from 52-70% and disease severity ranged from 2-5 but B. fabae isolated from Sinbellawen (B1) recorded the highest values of both disease incidence (70%) and disease severity (5).

Antagonistic effect of Trichoderma harzianum against B. fabae on solid medium )dual culture test): Data in Table 2 showed that in single culture plates, the linear growth of both B. fabae and T. harzianum increased by the time after inoculation. It was noticed that, the linear growth of T. harzianum was more rapid than that of B. fabae. In dual culture plates, a great inhibition to the growth of B. fabae was occurred after 6 days in comparison with control. Six days after inoculation, it was observed that T. harzianum overgrow B. fabae(Fig. 1 ) indicating the antagonistic behavior of T. harzianum against B. fabae.

Antifungal activity of the non-volatile metabolites of Trichoderma harzianum against B. fabae: A complete inhibition of the mycelial growth of B. fabae (except the growth on the disc itself) was observed compared with the control (Fig. 2), which indicated the antifungal activity of T. harzianum metabolites.

Table 1:	Pathogenicity test for the different isolates of Botrytis fabae
*Each value represents the mean of 3 replicates, **2: Small spots, 3: Increase in spots number and its spread, 4: Coalesce of spot together and 1/4 of leaf surface is necrotic and 5: 1/2 of leaf surface is necrotic

Table 2:	Antagonistic effect of Trichoderma harzianum against Botrytis fabae (dual culture test)
*Distance between the disc’s center and the margin of the colony, **Each value represents the mean of 5 replicates

Fig. 1(a-d):	Antagonistic effect of Trichoderma harzianum against B. fabae on solid medium after (a) 2 days (b) 4 days (c), 6 days and (d) 8 days

Scanning electron microscopy observations on the mycoparasitic nature of Trichoderma harzianum on B. fabae: Mycelial samples from the interaction region of dual culture of B. fabae and T. harzianum were observed in a scanning electron microscope, the events of mycoparasitism mechanism are shown in the scanning electron micrographs (Fig. 3). Hyphae of T. harzianum frequently grew parallel to the hyphae of the host (B. fabae) and sticks itself onto it’s surface (Fig. 3a), this process followed by rapid and excessive coiling and formation of appressoria-like structures on the host surface (Fig. 3b) and finally lysis of host cell walls was also observed (Fig. 3c), these observations indicated the mycoparasitic nature of T. harzianum on B. fabae.

Fig. 2:	Antifungal activity of the non-volatile metabolites of Trichoderma against B. fabae

Fig. 3(a-c):	Scanning electron micrograph showing hyphae of B. fabae ‘B’ hyphae of Trichoderma harzianum ‘T’ and appressoria like structures, ‘A’. (a) Trichoderma harzianum attach to the host and coil around it, (b) Form appressoria on the host surface and (c) Finally the host cell wall lysed

DISCUSSION

Since the chemical pesticides are the most commonly known methods for the management of fungal diseases in fields and greenhouses, some considerable problems threaten to limit the continued use of them. Firstly, some fungi develop resistance to a number of chemicals; secondly, some synthetic fungicides are not readily biodegradable and tend to persist for many years in the environment. The toxic effects of the fungicides on the non-target organisms may also have undesirable changes in the environment²⁰. Because of these associated problems, scientists are trying to test environmentally safe alternative means for the disease control, including the biological control methods²¹.

So that, this study was planned to replace the undesirable and unsafe chemical control by another effective, inexpensive and safe options for faba bean chocolate spot disease control. Trichoderma spp., specially T. harzianum were suggested to be used as a biocontrol agents for plant pathogens of faba bean plants^18,22.

The antagonistic effect of T. harzianum against B. fabae on solid medium was investigated using dual culture technique and it was observed that, the linear grow th of T. harzianum was more rapid than that of B. fabae. A great inhibition to the growth of B. fabae was clearly observed. An overgrowth of T. harzianum on B. fabae was observed 6 days after inoculation and dots of conidia are seen on the surface of the phytopathogen. The antagonistic mode of action of the fungus and the ability of conidia to germinate on phytopathogen’s surface has been proposed for the production of antibiotics⁹ and the fact that, they are already possess a set of cell wall degrading enzymes such as chitinases, glucanases and proteases²³.

The antifungal activity of T. harzianum metabolites was also tested against the linear growth of B. fabae using the cellophane method. A complete inhibition of the growth of B. fabae was observed indicating the antifungal activity of T. harzianum metabolites, these results were in agreement with the findings of Bennet and Lane²⁴ and Doi and Mori²⁵. This inhibition can be explained according to the generally accepted definition of antibiosis "The mechanism mediated by specific metabolites such as antibiotics, enzymes and non-volatile compounds²⁶”. Most Trichoderma strains produce volatile and non-volatile toxic metabolites that impede colonization by antagonized microorganisms among these metabolites, the production of harzianic acid, tricholin, peptaibols, viridian, heptelidic acid and antibiotics²⁷. The combination of hydrolytic enzymes and antibiotics results in a higher level of antagonism than that obtained by either mechanisms alone²⁸.

In the present study the mycoparasitic nature of T. harzianum on B. fabae was also investigated by the light microscope using slide culture method. It was observed that the two fungi grew toward each other and their hyphae intermingled with slight mycoparasitic behavior. Light microscopic observations revealed that there was evidence about the mycoparasitic nature of the tested isolate of T. harzianum on B. fabae. In this connection, the scanning electron microscopy observations confirmed that mycoparasitism appeared to contribute to the aggressive nature of the tested isolate of T. harzianum against B. fabae. These results are in good agreement with Monte and Liobell²⁹ reported that most isolates of the genus Trichoderma were found to act as mycoparasites of many economically important aerial and soil borne plant pathogens. Also, these observations were in agreement with that of Labudova and Gogorova⁸ and Harman⁹, in these studies the lytic action of the pathogen was clearly appearant and the inhibition of the growth appears directly related to the ability of T. harzianum to hydrolyze the cell walls of the tested microorganisms rather than through inhibitory action of the antibiotics or toxins, their results revealed that, the nature of T. harzianum antagonism is based on mycoparasitism (lysis) and appeared to optimize with contact between the mycelia.

In contrast, Elad et al.²³ found that, the mycoparasite T. harzianum was unable to degrade the cell wall of Fusarium oxysporum and that these hydrolases of Trichoderma were actively involved in the microbiological control. Also, Rashad³⁰ showed that there was no evidence about the mycoparasitic nature of T. harzianum on Bipolaris orayzae, this mean that the mycoparasitism phenomenon may be host specific. In all experiments dealing with Trichoderma spp., specificity of antagonist for a range of host fungi has been reported.

CONCLUSION

The above results indicated the antagonistic action of T. harzianum against B. fabae. Moreover, the antifungal activity of T. harzianum metabolites was also indicated. Light microscopic observations revealed that there was evidence about the mycoparasitic nature of the tested isolate of T. harzianum on B. fabae. In this connection, the scanning electron microscopy observations confirmed that mycoparasitism appeared to contribute to the aggressive nature of the tested isolate of T. harzianum against B. fabae. These results strongly recommend using the save biological control agent T. harzianum as an alternative to the harmful chemical fungisides to manage the phytopathogen B. fabae.