Volatile and Non-Volatile Compounds of Trichoderma

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.