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.


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)(8)(9)(10)(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 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.
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: 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.

٢٢
From the above it appears that the isolate T.harzianum is more efficient against the tested fungi than T. viride and that is in agreement with (3).
On the other hand, and from previous studies (data not shown), it is well known that some kinds of Gram Positive bacteria such as Bacillus subtilis and Bacillus cereus (32) and some kinds of Gram Negative bacteria such as Pseudomonas spp.(P.aeruginosa and P.flourescens) (33) have an antifungal activities against a variety of fungi, and when we test these bacteria (Bacillus subtilis and Pseudomonas aeruginosa) for their antifungal activity against T. harzianum and T.viride, they showed no effect on these fungi.
It is well known that some kinds of T.harzianum produce anovel tetramic acid with a remarkable activity against Gram-positive bacteria especially Staphylococcus aureus and Bacillus subtilis (34), but when our culture filtrates of T.harzianum and T.viride were tested against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, they showed no activity, also they showed no activity against the Gramnegative bacteria Pseudomonas aeruoginosa and E.coli.

Extraction and partial characterization of the antifungal compound(s) of T.harzianum and T. viride :
As mentioned by the method described by (33), the crude extract of each T.harzianum and T.viride (which was dissolved in butanol) was taken for serial dilutions and another extracts were taken and evaporated from butanol for further studies.
It was found that the optimum conditions for the production of antifugal compound(s) from T.harzianum and T.viride was growing the fungi at 28ºC for two weeks on potato dextrose agar (for antifungal assay) or potato dextrose broth for production and extraction of the antifungal compounds, and that was in agreement with (33).
The culture filtrate (not diluted) of T.harzianum showed an inhibition zone of 1.5 cm on Alternaria alternata and of 1 cm on Fusarium graminearum, while the culture filtrate of T. viride showed inhibition zone of 1cm on A.alternata and of 0.7 cm on Fusarium graminearum (Figure 2), but the culture filtrate of both T.harzianum and T.viride showed no effect against the fungi Aspergillus niger, Aspergillus flavus, and Penecillum nalgiovense .When the culture filtrate of both T.harzianum and T.viride was subjected to heat treatment (100˚C for 10 minutes), there was no effect on the activity of the compounds because they showed approximately the same results of the untreated compounds on A.alternata and F.graminearum.
Also, the culture filtrates showed solubility in organic compounds such as butanol, ethanol, dimethyl sulfoxide (DMSO) and others in addition to water.
When an FTIR analysis was done to the culture filtrate of T.harzianum and T.viride (Figure 3 and 4), they showed approximately the same absorbtion bands of the active compounds with a little difference in the absorbtion band of 1721.98 in the compound of T.viride.
Also the FTIR results showed the presence of an aromatic ring in both compounds of T.harzianum and T.viride.From all above it seems or we can conclude that the extracted antifungal compound(s) of both T.harzianum and T.viride had many similar characteristics with a little difference as demonstrated in the FTIR analysis that is recommended to do full characterizations and analysis of these compounds such as mass spectrophotometry, HPLC, and NMR etc., but it is most likely that they are from the same group of antibiotics that have an aromatic ring, but with the great difference in their action in which the fungus T.harzianum had much more antifungal activities than the fungus T.viride as seen in our research and also because of the greater inhibition zone of the culture filtrate of T.harzianum than the culture filtrate of T.viride.

Figure ( 1 )
Figure (1): A: The growth of the fungus Alternaria alternata (left) and growth of the fungus Fusarium graminearum(right) {Controls}.B:The growth of the fungus Trichoderma harzianum (left) and the overgrowth of the fungus T.harzianum over the fungi A.alternata, and F.graminearum.

Figure ( 2 ):
Figure (2): The effect of the culture filtrate of the fungus T.harzianum on the fungi A.alternata (left) and F.graminearum (right).

Figure ( 3 )
Figure (3): The FTIR analysis of the active compound(s) of the fungus Trichoderma harzianum.

Figure ( 4 )
Figure (4): The FTIR analysis of the active compound(s) of the fungus Trichoderma viride.