The antifungal activity of bacterial isolates of cassava products origin as well as a known antifungal agent Griseofulvin was determined. The fungi used were Aspergillus flavus, A. niger and Rhizoctonia solani. The antagonists were: Pseudomonas flourescens, Escherichia coli, Bacillus subtilis and B. pumilus. The results obtained showed that the degree of antagonistic activity against the fungal isolates varied. In vitro bioassay using the disc diffusion technique showed that Bacillus pumilus had considerable degree of antagonistic activity against Aspergillus flavus with MIC value of 2.0 while Escherichia coli had significant activity against Rhizoctonia solani (2.0) and Aspergillus flavus (4.0). The antagonistic activity of Pseudomonas flourescens against Aspergillus niger was observed within 24-48 h of growth. Varying degrees of activity were observed after incubation period using the known antifungal agent Griseofulvin against fungal isolates and its activity against Aspergillus niger far exceeded 48 h. The minimal inhibitory concentration obtained using the agar well diffusion technique and measured in percentage varied considerably. The least concentration for inhibition was 2.0 using bacterial metabolites while the antifungal agent was able to inhibit the growth of a fungus at concentration of 0.5. The metabolites exhibited stronger antifungal effect at higher concentration and the preliminary results of the investigation appears to indicate the suitability of bacteria isolates in the antagonism of fungal pathogens of cassava.
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The wide and indiscriminate use of chemical preservatives has been the cause of the appearance of resistant microorganisms over and over, leading to the occurrence of emerging food borne diseases (Gibbons, 1992; Kaur and Arora, 1999; Akinpelu, 2001). As a result of this, the interest to obtain alternative antimicrobial agents for use against food pathogens has increased. The possibility of controlling pathogenic fungi by antagonistic microorganisms has been explored by various workers (Blakeman, 1985; Mercier and Reelender, 1987; Pandey et al., 1993; Agarry, 2005). The development of antifungal drugs (agents) has lagged behind than that of antibacterial agents. This is a predictable consequence of the cellular structure of the organism.
Antagonism is the interference with or inhibition of the growth of a living organism due to the production of a specified antibiotic substance. An antagonist is an organism that adversely affects the growth of other microorganism growing in association with it. Antagonist is sometimes introduced to the surface of products with the aim of controlling a pathogenic organism of that product. This is referred to as biocontrol. Numerous antagonistic microorganisms with the potential for biocontrol of plant diseases has been reported and identified over the past few years (Fravel, 1988; Fereira et al., 1991).
Effective antifungal agents frequently either extract membrane sterols or prevent their synthesis (Lansing et al., 2005). Griseofulvin has been available since 1958 in treating diseases caused by fungi. Griseofulvin comes from the mould, Penicillium griseofulvum. It stops fungal cells division (i.e., fungistatic) but does not kill them outrightly (Gull and Triner, 1973).
This study was carried out to compare and contrast the antagonistic activity of bacterial isolates of cassava product origin and griseofulvin against the pathogenic fungi.
MATERIALS AND METHODS
Stock cultures of previously identified bacteria and fungi strains isolated from cassava products viz., grated cassava, starch and flour were used (Olowoyo et al., 2001).
Determination of Spore Count
The fungi namely Aspergillus flavus, A. niger and Rhizoctonia solani were cultured on potato dextrose agar plates for 7 days. The cells were separately harvested by washing with sterile 0.85% physiological saline into suitable receptacles. The cell suspension was then diluted with the same liquid and the spores counted using Hawksley haemocytometer with Neubauer ruling (Evandro et al., 2005).
Determination of Bacteria Population
The bacterial isolates were inoculated in broth and incubated at 37°C for 24 h. The broth cultures were then diluted to a dilution factor of 10-8. One milliliter of the broth culture was taken from the test tube with dilution factor of 10-8 and pour plated. The plates were incubated and after 24 h the colony forming unit per mL was counted.
Extraction of Metabolite
The 24 h broth culture was decanted aseptically into sterile centrifuge tubes at equal volumes. The tubes were then centrifuged for 20 min at 3000/rev/min. The supernatant containing the bacteria metabolite was prepared by adding 0.4 mL of the metabolite to 0.04 mL of Tween 80. The mixture was then made up to 5 mL by adding sterile distilled water and shaken vigorously for 5 min (Evandro et al., 2005). The stock of the antifungal agent griseofulvin was prepared by dissolving 500 g of griseofulvin in 0.4 mL of sterile distilled water and then completing the mixture to 5 mL by adding sterile distilled water. The percentage final concentration of both the metabolite and griseofulvin were achieved.
Screening of Antifungal Activity
In vitro test of the antagonistic activities of bacterial isolates on fungi was performed using a modified disc diffusion tests described by Lima et al. (1993). One milliliter of the fungal suspension prepared with sterile physiological saline solution was uniformly spread on acidified potato dextrose agar plates. After inoculums absorption, sterile filter paper discs (Whatmann No. 1 diameter 6 mm) were soaked with the bacterial metabolites and placed at different points on the potato dextrose agar plates inoculated with the fungal suspension. The plates were then incubated at 25-28°C for 5-7 days. At the end of the incubation period, the inhibition halo diameters were measured and expressed in millimeters. When the inhibition halo diameter observed was equal or higher than 10 mm diameter, it was considered a positive antifungal activity. The same procedure was followed while screening for the antifungal activity of griseofulvin.
Determination of the Minimum Inhibitory Concentration
Bacterial isolates that presented antifungal activity in the screening assay were evaluated for their minimum inhibitory concentration using the plate diffusion procedure involving wells in dishes (Hadeck and Gregor, 2000). This was achieved by uniformly spreading 1 mL of fungal suspension prepared with sterile 0.85% physiological saline solution on acidified Potato Dextrose Agar (PDA) plates. After inoculum absorption by acidified PDA, wells were made using sterile cock borers, which were then filled with 0.1 mL of the different percentage final concentration of the bacteria metabolite. The control was carried out by filling the wells with 0.1 mL of sterile distilled water. The same procedure was followed for the determination of the minimum inhibitory concentration (MIC) of the antifungal agent griseofulvin.
In vitro Bioassay of Bacterial Antagonists
Not all bacteria isolates were antagonistic. Three bacteria namely Bacillus pumilus, Pseudomonas fluorescens and Escherichia coli were able to inhibit the growth of three fungi namely; Aspergillus flavus, A. niger and Rhizoctonia solani. The minimum inhibitory concentrations of the bacterial metabolites were obtained using the agar well diffusion technique at different time intervals (Fig. 1-3). Bacillus pumilus had MIC value of 2.0 on Aspergillus flavus while Escherichia coli had MIC values of 2.0 and 4.0 on Aspergillus flavus and Rhizoctonia solani, respectively.
In vitro Bioassay of Griseofulvin
The minimum inhibitory concentrations obtained using the agar well diffusion bioassay technique at different time interval for the fungi are presented in Fig. 4-6. The MIC values of griseofulvin on Aspergillus flavus was 0.5, Aspergillus niger (2.0) and Rhizoctonia solani (1.0). Griseofulvin inhibited the growth of the fungal isolates at reduced concentration than that of the bacterial metabolites.
|Fig. 1:||Antagonistic activity of Bacillus pumilus on Aspergillus flavus at different time intervals|
|Fig. 2:||Antagonistic activity of Escherichia coli on Rhizoctonia solani at different time intervals|
|Fig. 3:||Antagonistic activity of Escherichia coli on Aspergillus flavus at different time intervals|
|Fig. 4:||Antagonistic effect of Griseofulvin on Aspergillus niger at different time intervals|
|Fig. 5:||Antagonistic effect of Griseofulvin on Rhizcotonia solani at different time intervals|
|Fig. 6:||Antagonistic effect of Grisofulvin on Aspergillus flavus at different time intervals|
The growth of the mould was slow and resulted in abnormal misshapen colony development, which was difficult to quantify. Studies have shown that Bacillus sp are able to inhibit the growth of some species of Aspergillus while Esherichia coli has been shown to inhibit the growth of A. flavus (Agarry et al., 2005). Also, susceptibility patterns were observed between Escherichia coli and Rhizoctonia solani while the antifungal activity of Pseudomonas fluorescens on Aspergillus niger was observed within 24-48 h of growth after which the inhibition halo diameter could not be observed. Previous research in which Pseudomonas flourescens has been used as an antagonist against Aspergillus sp. revealed weak inhibition (Agarry and Osho, 2005).
The MIC values determined using the agar well diffusion technique were recorded at different time intervals. The MIC values for Bacillus pumilus on Aspergillus flavus were 2.0 while the values of Escherichia coli on Rhizoctonia solani and A. flavus was 2.0 and 4.0, respectively. For the fast growing fungal isolates namely Aspergillus niger and Rhizoctonia solani, the values were recorded after 48 h of growth while for the slow growing fungus Aspergillus flavus, the inhibition halo diameter was measured and recorded after 72 h of growth. The inhibition halo diameter at varying percentage final concentration gradually reduced and the values consistently decreased until a point was reached where the inhibition halo diameter became static. This may be attributed to the fact that the rate of killing slows down and subsequently stops due to the survival of more resistant strains of the fungal isolates (Lansing et al., 2005). The significance of the values showing the extent of antifungal activity of the bacterial isolates was the function of the concentration of the bacterial metabolites.
When the test bacterial isolates were replaced with a known antifungal agent griseofulvin, the susceptibility of Aspergillus niger to the agent far exceeded 24 - 48 h, However, when the MIC was determined for the agent, the least value was 0.5 against Aspergillus niger while the highest was 2.0 for Rhizoctonia solani. Aspergillus flavus had a reduced MIC value of 1.0 as compared to when the bacterial metabolite was used. It was observed that the antifungal agent was more effective than the bacterial metabolites.
The bacterial isolates possess some degree of antifungal activity against fungal pathogens of cassava products. Further studies will be carried out to extract and purify the bioactive ingredients of the bacterial metabolites.
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