Aba bean (Vicia faba L.) is considered the most important food legume crops in Egypt. The economic importance of faba bean cultivation in the world could be explained by its high nutritional value of vitamins, protein, carbohydrates, and some other compounds. Thus, it is a rich available source of food for both human and animals (Sahile et al., 2011). In addition, it improves the soil fertility through nitrogen fixation. Therefore, improving the production of this crop is one of the objectives in agriculture in many countries (Boubekeur et al., 2012).
The total cultivated area with faba bean in Egypt during 2012 growing season reached about 192500 feddan with a total production of 1.561.175 ardab (ardab =155 kg) at the rate of 8.11 ardab/feddan (Food Legume Statics Dept., Field Crops Res. Instit., ARC.,2012). Faba bean is liable to be attacked by many foliar diseases. However, chocolate spot caused by Botrytis fabae Sard. and Botrytis cinerea Pers. are considered the major destructive diseases affecting the crop, causing serious damage to the plant and decrease of the yield production more than 50% (Nassib et al., 1991; Bouhassan et al., 2004), especially in the north and middle parts of the Delta in Egypt and several countries in the world (Nassib et al., 1991).
The use of chemical control against this disease sometimes gives good results. However, improper use of fungicides leads mostly to environmental pollution, disasters throughout the world and the phenomena of resistance to B. fabae and B. cinerea (Brewer and Larkin, 2005). Therefore, to overcome these difficulties, it is urgent to apply alternative safe efficient methods against this disease. Biological control is considered an important approach of agricultural biotechnology in recent years for controlling many fungal plant pathogens (Deshmukh et al., 2010). Trichoderma spp. are the most promising and effective bioagents against various plant pathogenic fungi (Fahim et al.,1989; Kumar and Mukerjii, 1996). Trichoderma as antagonist for controlling a wide range of microbes was well documented and demonstrated for more than seven decades ago but its use under field conditions came much later (Fahim et al., 1989; Chet et al., 1997) and their mechanism of mycoparasitism is much more complex, that is nutrient competition, hyperparasitism, antibiosis, space and cell wall degrading enzymes (Abd-El-Khair et al., 2010). Several researchers have reported biological control as an effective method for controlling chocolate spot to reduce the use of fungicides (Hanounik and Hassanein, 1986; Abd El Moiety et al., 1990; Elad and Zimand, 1992; Abou-Zeid et al., 2003; Mahmoud et al., 2012). It was also found that there is a large variety of volatile secondary metabolites produced by Trichoderma such as ethylene, carbon dioxide, hydrogen cyanide, aldehydes and ketones which play an important role in controlling many plant pathogens (Vey et al., 2001; Faheem et al., 2010; Nagendra and Kumar, 2011). growth in the range of 51.11 – 77.78%. T. album-2 gave the highest growth inhibition followed by T. harzianum-6 then T. harzianum-7 and T. hamatum-6 . This may due to the release of toxic metabolites into the medium (Akhtar, 1982), also it may be due to competition for space and nutrients with B. fabae, because the pathogen requires exogenous nutrients for germination and germ- tube elongation over a period of several hours on the phyllosphere before penetrating the host plant (Dubos and Bulit, 1981). Indeed, the confrontation test whether a direct way of culture medium or remotely showed inhibition of growth and sporulation of the pathogen. If there is contact between T. album and T. harzianum, the pathogen, Trichoderma colonies invade those B. fabae.
When the pathogen is confronted in a direct way of culture medium, Trichoderma spp. breakdown the mycelium and cause a reduction of sporulation on the edge of the zone of inhibition due to the secretion of antibiotic substances circulating in the culture medium. Saber et al. (2009) reported that all of the tested fungal antagonists showed reasonably higher growth rate than the pathogen B. fabae, which has a daily growth rate in the range of 15 – 35 mm/day. These might be producing antibiotics or extracellular enzymes, which inhibited the growth of the pathogen. Results indicated that spraying faba bean plants 24 h before inoculation with the tested pathogen with any of the tested Trichoderma spp. and Bio-Zeid as a fungicide significantly reduced chocolate spot severity compared with Bio-Zeid and control. T. album-2 was the high antagonistic isolate followed by T. harizianum-6, T. hamatum-6 and T. viride-2. The biomass of the pathogen was reduced in the presence of Trichoderma spp.
This may be due to an effect on germ-tube elongation and to a lesser extension of germination rate (Zimand et al., 1996). Trichoderma spp. are known to control pathogens either indirectly by competing for nutrients and space, modifying the environmental conditions, or promoting plant growth and enhancing plant defensive mechanisms and antibiosis, or directly by inhibition of growth and sporulation of the pathogen mechanisms such as mycoparasitism and enzyme production (Zimand et al., 1994; Bouhassan et al., 2004). The earlier studies also revealed that antimicrobial metabolites produced by Trichoderma spp. are effective against a wide range of phytopathogenic fungi, e.g. B. fabae, Fusarium oxysporum, Rhizoctonia solani, Curvularia lunata, Bipolaris sorokiniana and Colletotrichum lagenarium (Fahim et al., 1989; Svetlana et al., 2010). The obtained results showed that, T. album-2 exhibited maximum growth inhibition to the mycelial growth of B. fabae followed by T. harzianum-6 compared to the control. This inhibition was more pronounced in the case of T. album-2. It appears that despite the absence of direct contact between Trichoderma spp. and isolates of B. fabae, the first may have an inhibitory activity on the development of colonies of B. fabae.
This could be explained by the ability of Trichoderma spp. to produce volatile substances that are able to limit and even stop the development of the pathogen. Also it is found that there is large variety of volatile secondary metabolites produced by Trichoderma such as ethylene, carbon dioxide, hydrogen cyanide, aldehydes and ketones, which play an important role in controlling the plant pathogens (Vey et al., 2001; Faheem et al., 2010; Nagendra and Kumar, 2011). The volatile substances caused significant inhibition to the sporulation of the causal fungus compared to the control. These observations suggested the possibility of secretion of antagonistic substances that diffuse into the culture medium, which cause lysis of the mycelium and spores of the pathogen.
The non-volatile secondary metabolites of Trichoderma species were found more effective in suppressing the mycelial growth of B. fabae. T. album-2 exhibited the highest effect on the mycelial growth of B. fabae followed by T. harzianum-6. After 7 days of confrontation, the colonies of Trichoderma spp. completely overlapped and covered the colonies of the B. fabae due to their mycoparasitism. Elad et al. (1993) described the action of T. harzianum and T. hamatum on B. fabae. Trichoderma spp. attacks the host by winding the mycelium around the host hyphae. Subsequently, the mycoparasite penetrates the host cells and uses the cytoplasmic contents. In this respect, culture filtrates of T. album-2 was the most effective in reducing the linear growth of B. fabae followed by T. harziunum-6 compared with control treatment. The non-volatile secondary metabolites from Trichoderma species (T. album-2 and T. harziunum-6), were found more effective in suppressing the mycelial growth of B. fabae when compared to volatile compounds.
In this respect, culture filtrate of T. album-2 was the most effective in reducing the linear growth of B. fabae followed by T. harziunum-6 in this respect. This study showed that there were promising antagonistic species of fungi prevalent on faba bean leaves, which can be exploited for the control of chocolate spot. The genus Trichoderma comprises a great number of fungal strains that act as biological control agents for the control of plant diseases and for their ability to increase plant growth, the antagonistic properties of which are based on the activation of multiple mechanisms. The antagonistic nature may be due to antibiosis, nutrient competition and cell wall degrading enzymes. The present study clearly showed the effect of the two antagonistic strains of Trichoderma isolates (T. album and T. harzianum), against B. fabae. Volatile and non-volatiles compounds produced by selected Trichoderma spp. drastically reduced the mycelium growth and spore production of B. fabae in-vitro. Based on the present investigation a new strategy will be developed for controlling chocolate spot disease on faba bean in vivo.