Macroscopically, the Aspergillus flavus colonies isolated from the maize grains showed rapid growth and appeared dark on upper side (surface) of the plate and creamy on its lower side (reverse). The texture was cottony and granular in appearance. Microscopically, the conidiophores were long, colourless and contained phialides. At the end of the conidiophore was borne globose conidia (Fig. 2).
Pathogenicity test of Aspergillus flavus
Maize inoculated with spore suspension of Aspergillus flavus started to rot after 4 days of incubation. The inoculated grains were totally consumed with extensive mycelial growth that formed dark coloration over the testa of the grains (Fig. 3). Comparison of the symptoms that emerged from the inoculated kernels to those of the control (no spore inoculation) confirmed the pathogenicity of the A. flavus isolates on the grains.
Antifungal activity of Ocimum kilimandscharicum oil on Aspergillus flavus by Agar dilution method
Results showed that the Ocimum kilimandscharicum oil inhibited the radial growth of Aspergillus flavus. The magnitude of the growth inhibition of the oil varied with its concentrations. Radial growth inhibition was 100% (no further growth of A. flavus occurred) at high concentrations (26.67, 20, 13.33, and 6.67) μl/ml. In the control experiment (plain PDA), a rapid growth of Aspergillus flavus was observed (Fig. 4).
Figure 4. Amended PDA plates with varied concentration of Ocimum kilimandscharicum oil showing Aspergillus flavus colony.
Analysis of variance showed no significant difference of radial growth inhibition amongst the oil concentration level of 26.67, 20, 13.33, and 6.67 μl/ml. The oil concentration level of 3.33 μl/ml resulted in 43% radial growth inhibition. Analysis showed a significant difference (p < 0.0001) in the growth inhibitory effect between the 3.33 μl/ml concentration level and plain PDA (Table 1).
Table 1. Mean colony diameter (in millimetres) of A. flavus on PDA amended with different concentration of O. kilimandscharicum oil
Mean Colony diameter (mm) Concentration (μl/ml) Plain PDA (control) 1.36±0.05a 3.33 1.17±0.30b 6.67 0.79±0.001c 13.33 0.79±0.001c 20 0.79±0.001c 26.67 0.79±0.001c Days Day 1 0.79±0.001d Day 3 0.85±0.20c Day 5 0.91±0.50b Day 7 0.93±0.60a P-values Concentration 0.0001 Day 0.0001 Concentration*Day 0.0001 Values expressed a r e means ± SEM for four replicates. Means within respect columns accompanied by similar lower-case letters do not differ significantly at P < 0.0001
A significant difference (p < 0.0001) in colony diameter of Aspergillus flavus was also observed between the days (day 1, day 2, day 3, day 5 and day 7).
Furthermore, a significant interactive effect (p < 0.0001) was observed between concentrations of the oil and days of colony diameter (Fig. 5).
Figure 5. Interaction effects of concentration of Ocimum kilimandscharicum oil and days of incubation on colony diameter.
Antifungal activity of Ocimum kilimandscharicum oil on Aspergillus flavus by disc diffusion
Just as the agar disc diffusion method, similar radial inhibition pattern was observed using the disc diffusion method. Radial zones of inhibition recorded after 7 days for the different concentrations were as follows; 400 μl/ml (radial inhibition = 7.08 mm), 300 μl/ml (radial inhibition = 6.53 mm), 200 μl/ml (radial inhibition = 6.10 mm), 100 μl/ml (radial inhibition = 5.93 mm) and 50 μl/ml (radial inhibition = 5.05 mm).
Analysis of variance showed that a highest zone of inhibition (p < 0.0001) occurred at concentration level of 400 μl/ml. There was also a significant difference between the treatment concentrations and the control experiment (p < 0.0001). Furthermore, there was a significant difference in the zone of inhibitions between the days of incubation (day1, day 3, day 5 and day 7).
A significant interactive effect (p < 0.0001) was also observed between the concentrations of the oil and days on colony diameter (Fig. 6).
Table 2. Mean zone of inhibition (mm) of Aspergillus flavus culture grown on filter paper discs impregnated with Ocimum kilimandscharicum oil
Size of zone of inhibition Concentration (μl/ml) Plain PDA (control) 5.00±0.00f 50 5.06±0.01e 100 5.58±0.09d 200 5.76±0.12c 300 5.98±0.16b 400 6.34±0.22a Days Day 1 5.00±0.00c Day 3 5.74±0.09b Day 5 6.11±0.15a Day 7 6.14±0.16a P-values Concentration 0.0001 Day 0.0001 Concentration*Day 0.0001 Values expressed are means ± SEM for four replicates. Means within respect columns accompanied by similar lower-case letters do not differ significantly at P <0.0001 by Tukey's HSD test.
Identification of Aspergillus flavus from maize
On behalf of the other authors, I (Corresponding Author) confirm that the manuscript has been read and is approved for submission
|Kaguchia SM, Birgen JK, Lang'at-Thoruwa C, Akwa TE. 2021. Antifungal effects of Ocimum Kilimandscharicum oil on Aspergillus flavus Infecting Maize after Harvest. Studies in Fungi. 6: doi: 10.5943/sif/6/1/19|