Fungitoxicity of Trichoderma longibrachiatum (Rifai) Metabolites against Fusarium oxysporum , Aspergillus niger and Aspergillus tamarii

An experiment was carried out to evaluate the fungitoxic effect of Trichoderma longibrachiatum (Rifai) metabolite on F. oxysporum, A. niger and A. tamarii. The fungi were collected from the International Institute of Tropical Agriculture (IITA) and Nigerian Institute of Science Laboratory Technology (NISLT). T. longibrachiatum was cultured on 1/4 strength Potato Dextrose Broth (PDB) following standard procedures. Its metabolite was extracted using 50 ml n-hexane with 50 ml Potato Dextrose Broth (PDB). The metabolite was purified by filter sterilization using a sterile 0.22 millipore filter disc after centri-fuging at 900 rpm for 20 minutes. Petri plates of each fungus were later im-pregnated in triplicates with the T. longibrachiatum metabolites using four concentrations (10%, 25%, 50% and 100%), and three volumes (1 ml, 2 ml, and 3 ml). Petri plates of fungi without the metabolite and 25% concentrations was better than that at 10% concentration of the metabolite. Trichoderma longibrachiatum metabolite can thus be said to possess promising fungitoxic potential even at concentrations as low as 25%. Both PDB and n-hexane are good extraction media for the metabolite.


Introduction
On a yearly basis, a huge percentage of economic plants are destroyed by plant pathogens. Pesticides and organic compounds are widely used to control plant pathogens in many countries. However, apart from the high cost, degradation of such compounds is very difficult and the concentration and/or accumulation of them in food chains continue to lead to higher toxicity levels in man and animals, as well as the environment [1] [2]. There is therefore an increasing focus on alternate methods for the control of plant diseases [3] [4]. Fusarium oxysporum is a pathogen documented to be the cause of several diseases in different crops [5] [6]. Aspergillus niger and A. tamarii are also known to be amongst the Aspergillus species causing common postharvest diseases in different plants [7]. Some strains of A. niger have been reported to produce potent mycotoxins called ochratoxins.
Trichoderma species are fungi that are present in nearly all soils and other diverse habitats. In soil, they frequently are the most prevalent culturable fungi.
They are favoured by the presence of high levels of plant roots, which they colonize readily. Some strains are highly rhizosphere competent, i.e. able to colonize and grow on roots as they develop. In addition to colonizing roots, Trichoderma species attack, parasitize and otherwise gain nutrition from other fungi. They are known worldwide as biological agents used in protecting crops against plant pathogens under greenhouse and field conditions. Their mode of action against plant pathogens is known to include antibiosis, mycoparasitism, competition for space and nutrients etc. [ Trichoderma species are highly efficient producers of many extracellular enzymes. They are known to produce different volatile organic compounds [16] [17].
The metabolites secreted by them are as well known to be used as bio-fungicides to fight plant diseases caused by pathogenic fungi [18]- [23]. The experiment was set up to investigate the fungitoxicity of Trichoderma longibrachiatum metabolite against the growth of Fusarium oxysporum, Aspergillus niger and Aspergil-

Collection of Fungal Isolates
Cultures of Trichoderma longibrachiatum, Fusarium oxysporum, Aspergillus niger and Aspergillus tamarii were collected from the International Institute of Tropical Agriculture (IITA), Ibadan.

Extraction of Metabolites from T. longibrachiatum
The metabolite of T. longibrachiatum was extracted using n-hexane and 1/4 strength Potato Dextrose Broth (PDB). A 5 mm cork borer was used to cut 10 mycelia discs from 7-day old cultures of T. longibrachiatum. A modified method of Spiegel and Chet [18] was used. T. longibrachiatum disc was transferred into each 250 ml flasks containing 100 ml of 1/4 strength of Potato Dextrose Broth (PDB), 50% n-hexane and 50% PDB. This was incubated and agitated on the horizontal shaker for 7 days at room temperature. After seven days of incubation and shaking, spores and mycelia of the fungi were removed from the broth culture of 1/4 strength Potato Dextrose Broth, 50% n-hexane and 50% PDB, by filtration using Whatman No. 1 filter paper. They were further purified by centrifuging at 9000 rpm for 20 mins and then filter sterilized using a sterile 0.22 millipore filter disc. The antimicrobial potential of the extracted metabolite was then tested against the three selected fungi at various concentrations of 10%, 25%, 50% and 100%.

Impregnating Test Plates with Trichoderma Metabolites and Data Collection
Acidified Potato Dextrose Agar (APDA) was prepared and dispensed into several Petri plates. Thereafter, 1 ml, 2 ml, and 3 ml of the metabolite were mixed separately with the Potato Dextrose Agar in the sterile plates by gently swirling plates to ensure homogeneity. The APDA plates with no metabolite served as a control. The plates were left to cool and gel, after which 5 mm fungal disc of each of the three pathogens was inoculated separately on the Agar plates under sterile conditions. The plates were incubated at 28˚C for 7 days and radial and diametric growth were measured daily. All experiments were done in triplicates.

Data Collection and Analysis
Percentage inhibition of pathogen was calculated by the following formula: where: L = Inhibition of mycelia growth; R 1 = Growth measurement of the pathogen in control; R 2 = Growth of the pathogen in the presence of Trichoderma longibrachiatum  [24].
Results obtained were subjected to analysis of variance (ANOVA) at the National Centre for Genetic Resources and Biotechnology (NACGRAB) using the Generalized Linear Model option of SAS (version 9.3). (P > 0.0001) was also highly significant. Table 2 gives the comparison of effectiveness of the metabolite of T. longibrachiatum at different concentrations against the growth of isolated pathogens. For F. oxysporum, growth inhibition of the pathogen at all the concentrations of the metabolite differed significantly from the control. Growth inhibition at 100% concentration was significantly higher than that at other concentrations. Growth inhibition at 25% concentration was also significantly higher than that at 50% concentration, which in turn, was significantly higher than that at 10% concentration (P = 0.05, R 2 = 0.99). For A. niger, growth inhibition of the fungus at all the concentrations were significantly higher than control. However, growth inhibition at 10% concentration was significantly lower than that in other concentrations (P = 0.05, R 2 = 0.99), which were not significantly different from themselves. For A. tamarii, growth inhibition of the fungus at all the concentrations was significantly higher than control. Growth inhibition at 25% concentration was significantly higher than that in other concentrations. Growth inhibition at 10% concentration was also significantly higher than that in 50% and 100% concentrations, while that at 50% concentration was as well significantly higher than that at 100% concentration (P = 0.05, R 2 = 0.99). Table 3 compares the growth inhibition of the isolated fungi (Fusarium oxysporum, Aspergillus niger and A. tamarii,) by Trichoderma longibrachiatum metabolite among days of incubation. Growth inhibition at all days of incubation was significantly higher than control (P = 0.05, R 2 = 0.99).  Table 4 compares effectiveness of T. longibrachiatum metabolite at different volumes on growth of the isolated fungi (F. oxysporum, Aspergillus niger and Aspergillus tamarii.). Growth inhibition of the three pathogens at the three different volumes was significantly higher than control. For F. oxysporum, growth inhibition at 3 ml of the metabolite was significantly higher than that at 2 ml, which in turn was as well significantly higher than that at 1ml of the metabolite.

Results
For Aspergillus niger, growth inhibition at 2 ml of the metabolite was significantly higher than that at 3 ml, which in turn was as well significantly higher than that at 1 ml of the metabolite (P = 0.05, R 2 = 0.99). For Aspergillus tamarii, growth inhibition at 3 ml of the metabolite was also significantly higher than that at 2 ml, which in turn was significantly higher than that at 1 ml of the metabolite (P = 0.05, R 2 = 0.99). Table 5 compares the growth inhibition of the three pathogens by metabolites of T. longibrachiatum extracted with different   solvents. Growth inhibition of the three pathogens by metabolites extracted with both solvents (n-hexane and PDB) was significantly higher than control. For both F. oxysporum and Aspergillus niger, growth inhibition by metabolite extracted with PDB was significantly higher than that extracted with n-hexane. However, growth inhibition of Aspergillus tamarii by metabolite extracted with n-hexane was also significantly higher than that by metabolite extracted with PDB (P = 0.05, R 2 = 0.99).

Discussion
The   [25] and Vicaino et al. [26]. Rifai [12] also wrote extensively on the biocontrol potential of Trichoderma species. The results obtained with metabolites extracted with both solvents compared to control (P = 0.05, R 2 = 0.99) showed the appropriateness of the two solvents (n-hexane and PDB) as extraction media for the Trichoderma metabolite. Both of them could thus be said to be good extraction solvents for T. longibrachiatum metabolite. The better result obtained with metabolite extracted with n-hexane might not be unconnected with its organic nature [18] [27]. Gams and Bissette [28] reported the degree to which Trichoderma species suppressed the growth of peanut moulds such as Fusarium species, A. niger, A. flavus, A. parasiticus and A. ochraceous might involve the production of volatiles and/or production of extracellular enzymes. Volatiles from Trichoderma species have been reported to be able to arrest the hyphal growth of different fungal pathogens on agar plates. Most strains of Trichoderma are reported to possess the potential to produce volatiles and non-volatile toxic metabolites such as harzianic acid, alamethicins and tricholin that hinder growth of other microorganisms [29]. Some aggressive Trichoderma spp. used as biocontrol agents are also known to be capable of producing either antibiotics or extracellular enzymes or both.

Conclusion
Conclusively, the metabolite of Trichoderma longibrachiatum could be said to possess promising antagonistic potential against Fusarium oxysporum, Aspergillus niger and A. tamarii in that order. Generally, the effectiveness of the metabolite is better at 100% and 50% concentrations and at a volume of 3 ml. Both PDB and n-hexane are good extraction solvents for Trichoderma longibrachiatum metabolite. Further studies are however necessary to ascertain its effectiveness in vivo over a long duration.