Antagonistic Activity and Production of Antifungal Compound(s) from Selected Trichoderma spp.

Two Trichoderma spp. (T.harzianum and T.viride) which are well known for their use as biocontrol agents were used in this study and tested for their antagonistic activity against several plant pathogenic (and even human pathogenic) fungi, also the activity was tested against selected Gram positive and Gram negative bacteria. The proper conditions for the production of the antifungal compound were determined, and after the production and extraction of the antifungal compounds from the two Trichoderma spp., an attempt to partially characterize the nature of the antifungal compound(s) was done using several chemical analysis methods, and the results showed that the inhibition was due to the production of antifungal compounds that had many similar characteristics for both T.harzianum and T. viride but with a difference in their antifungal actions. Antagonistic Activity and Production of Antifungal Compound(s) from ... 19 Introduction At present, around 30% of all plant species have been destroyed by plant pathogens. Pesticides and organic compounds are widely used to control plant pathogens in many countries. However, the degradation of such compounds is very difficult and the concentration and/or accumulation of them in food chains are leading to higher toxicity levels in animals (1). Trichoderma species have been investigated for over 80 years. They have been used recently as biological control agents and their isolates have become commercially available of late. This development is largely the result of a change in public attitude towards the use of chemical pesticides and fumigates such as methyl bromide (2 and 3). In this respect, Trichoderma spp. have been studied as biological control agents against soil-borne plant pathogenic fungi (4,5,6). Results from different studies showed that several strains of Trichoderma had a significant reducing effect on plant diseases caused by pathogens such as Rhizoctonia solani, Sclerotium rolfsii, Phythium aphanidermatium, Fusarium oxysporum, F. culmorum and Gaeumannomyces graminis var. tritici under greenhouse and field conditions (7-11). Knowledge concerning the behaviour of these fungi as antagonists is essential for their effective use because they can act against pathogens in several ways (12). Isolates of Trichoderma harzianum can produce lytic enzymes(13) and antifungal antibiotics (14,15,16) and they can also be competitors of fungal pathogens (10), and promote plant growth (9). Also isolates of Trichoderma viride are well known to produce antifungal metabolites like acetaldehyde. It was reported that the production of metabolites from different Trichoderma strains depends on ecological factors, and so the strains show varying effects on pathogens (17and 18). Some of these metabolites have been isolated from sporulating or mycelial cultures but subcultivation decreased the production of the peptide antibiotics produced by Trichoderma isolates (19and 20). The most frequently suggested mechanisms of biocontrol by Trichoderma include mycoparasitism, antibiosis, competition for nutrients, or all of the above. Trichoderma can inhibit the pathogen by means of antibiotics (21,22) or cell wall-degrading enzymes (CWDEs) (23) such as chitinases (24,25), glucanases (26), proteases (27), mannanases, and other hydrolases (28). The relative importance of these two mechanisms in the antagonistic process depends on specific pathogen host interactions (29,30). The aim of this study was to test and compare the biological activity of Trichoderma harzianum and Trichoderma viride against several plant (and even human pathogenic) fungi (also to test the antibacterial activity) and to produce and extract the active compound(s) from each fungus, and also to partially identify the nature of these compound(s). Materials and Methods Fungal strains : a) The biocontrol Trichoderma harzianum and Trichoderma viride that were used for the antagonistic activity were taken from the Plant Protection Department/College of Agriculture/Mosul University. Omar Mu'ayad Al-Obaidy & Maha Akram Al-Rijabo ٢٠ b) The plant pathogenic fungi Penicillium nalgiovense, Fusarium graminearum, Alternaria alternata, were taken from Biology Department/ College of Science/ Mosul University. c) The human pathogenic fungi Aspergillus niger, and Aspergillus flavus were taken from Biology Department/ College of Science/ Mosul University. Antifungal bioassay test ( Dual culture tests): Interactions between antagonistic fungi and pathogenic fungi were determined by the method described by Kucuk and Kivanc (35). Mycelial disks (7mm in diameter) of pathogenic fungi were placed on one edge of a petri dish containg potato dextrose agar (PDA) or Sabroud agar in the case of human pathogenic fungi, while mycelia disks of Trichoderma spp. (T.harzianum and T.viride) were placed on the opposite side of the plate. After the desired incubation time (about 7 days), at 28oC, the overgrowth of colonies of the test fungi by the antagonist was determined. Production and extraction of the antifungal compound(s) from Trichoderma spp. A water-soluble component(s) was extracted with butanol from the culture filtrates of T. viride and T.harzianum grown for 14 days. Pooled culture filtrate (50 ml) was clarified through Whatman no. 3 filter paper and extracted 2 times with 50 ml of butanol. The butanol extract (100 ml) were pooled dried in a water bath (22 liters Genlab limited) at 60°C for 7 to 8 h each day for three successive days. To determine the biological activity of the butanol extracts, the residues were dissolved each in 10 ml of sterile distilled water and from these solutions, 1 ml portion of each of the Trichoderma species extracts (100%) and the serial dilutions (70%, 50%, 30% and 10% if needed) was pipetted into different wells (8 mm diameter) borne on the surface of solidified PDA plates (9 cm diameter) previously prepared to allow excess water evaporate (pH6) (31). Partial characterization of the produced antifungal compounds: Several tests were done to partially characterize the nature of the active crude extract such as heat treatment, nature of the Compound(s), solubility of the compound(s), FTIR analysis and others. Results and Discussion: Antifungal bioassay test: The two Trichoderma spp. (Trichoderma harzianum and Trichoderma viride) were subjected to the bioassay test against different phytopathogenic (and even human pathogenic) fungi using the dual culture technique and the results were as follow: Antagonistic Activity and Production of Antifungal Compound(s) from ... 21 When the isolate Trichoderma harzianum grown with the fungus Alternaria alternata, the fungus T.harzianum completely cover the plate (over growth upon the fungus Alternaria alternata) (Figure 1), which mean that the fungus T.harzianum degrade or hydrolyse the fungus A.alternata and the same thing happened when T.harzianum grown with Fusarium graminearum and Penicillium nalgiovense and this may be due to the production of hydrolytic enzymes or compounds that completely hydrolyse the other fungi, but it has no effect on the fungus Aspergillus niger and Aspergillus flavus, while the fungus T. viride inhibits the growth (mycelial growth) of A. alternata in a ratio of 94.2 % and of F. graminearum in a ratio of 76.5% and have no effect on the growth of P. nalgiovense, Aspergillus niger, and Aspergillus flavus.