Team:NCTU Formosa/Fungal Result
Inhibition Zone Result
Sample
Each peptide was repeated the experiments for three times to ensure the results.
The HEPES buffer, which has no toxic on cells, is applied to be the negative control.
Peptide vs. Concentration
Peptide Observation
Figure 不知道是多少: The white part is mycelium, the holes were added different concentrations of peptides and negative control HEPES respectively.
If the peptides can inhibit the mycelium, then there will be a range around the holes that the mycelium can not occupied. Otherwise, the mycelium will grow ignoring the holes, like the hole containing HEPES. The whole scene will look like a white circle, while the edges of effective peptides concave.
- Brevinin-1E
Table 1: The result of inhibition zone with the different concentration of Brevinin-1E
Figure: The result of inhibition zone which used the peptide - Brevinin-1E
Figure: The comparison of partial magnification of the Figure 上一個
- T-SN2
Table : The result of inhibition zone with the different concentration of Temporin-SN2
Figure: The result of inhibition zone which used the peptide - Temporin-SN2
Figure: TThe comparison of partial magnification of the Figure 是一個
- Mastoparan-C
Table : The result of inhibition zone with the different concentration of Mastoparan-C
Figure: The result of inhibition zone which used the peptide - Mastoparan-C
Figure: The comparison of partial magnification of the Figureg上一個
- Pelophylaxin-4
Table : The result of inhibition zone with the different concentration of Pelophylaxin-4
Figure: 1095.6 Da venom vasodilator peptide
Figure: The result of inhibition zone with the different concentration of 1095.6 Da venom vasodilator peptide
The result of inhibition zone which used the peptide -1095.6 Da venom vasodilator peptide
- Caeridin-1.4
Table : he result of inhibition zone with the different concentration of Caeridin-1.4
Figure: The result of inhibition zone which used the peptide -Caeridin-1.4
Conclusion
According to the result, the Brevinin-1E and Temporin-SN2 were the strongest in the peptides we have selected, since their effectiveness on the mycelia were the most obvious. Mastoparan-C was not obviously, but still had effectiveness. 1095.6 Da venom vasodilator peptide, Pelophylaxin-4 and Caeridin-1.4 seemed to be no effect on the mycelia of Botrytis cinerea.
Spore Germination Result
Sample
Table:
Peptide vs. Concentration
Figure:
Calculation of Spore Germination
Figure:
Figure:
Observation
In the comparison with the inhibition zone and botany experiments, the spore germination was much hard to visualize the results only by the taken pictures. Because of the different lengths of germination tubes in one scene, what we would observe was the trend and tendency among different concentration and lengths.
Figure:
The spores were classified into four grades according to the lengths of the germination tubes, which were spores that did not germinate, the lengths of germination tubes were same as the spore itself, germination tubes three times longer than the spores and germination tubes five times longer than the spores. And then we calculated the percentage of each germination grade of each sample.
- Analysis of Spore Germination Tendency
Figure: In this bar chart above shows a comparison of spore germination between six kinds of peptides with a concentration of 1000ug/ml and the negative control.
We calculated the percentage of each spore level that we mentioned above and turned it into this bar chart. With this chart, we were able to know that after 6 hours of incubation, the spore germination percentage of non-germinating spores adding peptides with a concentration of 1000ug/ml was higher than adding the negative control, which meant the spore germination was inhibited by peptides and proved the peptides are effective.
Figure: In this bar chart shows a comparison of spore germination between six kinds of peptides with a concentration of 500ug/ml and the negative control
Except Pe-4, the other peptides had a better effectiveness than our control.
Comparing the result to the 1000ug/ml chart, we understood that we would need a higher concentration to inhibit Botrytis cinerea.
Figure: In this bar chart shows a comparison of spore germination between six kinds of peptides with a concentration of 250ug/ml and the negative control
Except Pe-4, the other peptides had a better effectiveness than our control.
Conclusion
Botany Experiment Result
Sample
Table:
Peptide vs. Concentration
Table: This table shows all the result of plant experiment
In order to test our antifungal peptides, we put peptides and the negative control on the flower. The negative control was on the left hand which was infected with pathogen and our peptide sprayed on the right.
Peptide Observation
Figure: Left: Botrytis cinerea infection on Oncidium petal; Right: uninfected Oncidium petal
- T-SN2
Figure: Left: Pathogens mixed with the negative control; Right: Pathogen mixed with Temporin-SN2
This picture shows that T-SN2 has the ability to inhibit fungi growth because the control which in the left side was obviously infected with pathogens and the right side wasn’t.
- B-1E
Figure: Left: Pathogens mixed with the negative control; Right: Pathogens mixed with Brevinin-1E
This picture shows that B-1E has the ability to inhibit fungi growth because the control which in the left side was obviously infected with pathogens and the right side wasn’t.
- 1095.6
Figure: Left: Pathogens mixed with the negative control; Right: Pathogens mixed with 1095.6 Da venom vasodilator peptide.
The black spot in this picture formed a strong contrast with the left side so we can say that 1095.6 Da venom vasodilator peptide has antifungal mechanism.
- Pe-4
Figure: Left: Pathogens mixed with the negative control; Right: Pathogens mixed with Pelophylaxin-4.
Although the right side of flower had a little bit infected, the left side was more obvious. According to the picture, we can presume that the peptides have antifungal ability.
- M-C
Figure: Left: Pathogens mixed with the negative control; Right: Pathogens mixed with Mastoparan-C.
The black spots in this picture were not very clear to see but in the right side of the flower there were three little spots on it. By these three little spots, we can say that this peptide ability to inhibit fungi from multiply.
Conclusion
According to our experiments, these peptides all have shown their antifungal characteristics.
Table:
Reference
