Team:NCTU Formosa/Fungal Result
Inhibition Zone Result
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.
Sample
Table 1: The peptides which we used to conduct the experiments
Peptide vs. Concentration
Table 2:The table shows all the results of the inhibition zone.
Peptide Observation
Figure 1: 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 3: The result of inhibition zone with the different concentrations of Brevinin-1E
Figure 2: The result of inhibition zone which used the peptide - Brevinin-1E
Figure 3: The comparison of partial magnification of the Figure 2
- T-SN2
Table 4: The result of inhibition zone with the different concentrations of Temporin-SN2
Figure 4: The result of inhibition zone which used the peptide - Temporin-SN2
Figure 5: The comparison of partial magnification of the Figure 4
- Mastoparan-C
Table 5: The result of inhibition zone with the different concentrations of Mastoparan-C
Figure 6: The result of inhibition zone which used the peptide - Mastoparan-C
Figure 7: The comparison of partial magnification of the Figure 6
- Pelophylaxin-4
Table 6: The result of inhibition zone with the different concentrations of Pelophylaxin-4
Figure 8: The result of inhibition zone with the different concentrations of Pelophylaxin-4
- 1095.6 Da venom vasodilator peptide
Table 7: The result of inhibition zone with the different concentrations of 1095.6 Da venom vasodilator peptide
Figure 9: The result of inhibition zone which used the peptide -1095.6 Da venom vasodilator peptide
- Caeridin-1.4
Table 8: The result of inhibition zone with the different concentrations of Caeridin-1.4
Figure 10: 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.
Table 9: The results of different concentration of peptides(circle: effective; triangle: less effective; X: ineffective)
Spore Germination Result
Sample
Table 9: Peptides and their sequences
Peptide vs. Concentration
Table 10: Experimental results
Calculation of Spore Germination
Draw some spore suspension to a double concave slide and observe it under a microscope. When observing the sample, we put a plastic sheet that has small knife scratches we scratched beforehand to divide the whole sight under the slide, and made sure that we didn’t choose the same sight . The plastic sheet divides the whole sight of the microscope into equal squares, and we picked the square relatively of number 1,3,5,7,9 and counted the number of spores.
Figure 11: We divided our sight into nine squares under eyepiece 10X, objective 4X.
Figure 12: The picture shows the squares for counting spores under eyepiece 20X, objective 4X
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.
Table 11: Illustration of four grades of spores according to the lengths of the germination tubes
The spores were classified into four grades according to the lengths of the germination tubes, 0 means the spores that did not germinate; 1, the lengths of germination tubes were same as the spore itself; 3 represents the germination tubes three times longer than the spores and 5 stands for the 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 13: 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 14: 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 for Pe-4, the other peptides had a better effectiveness than our control. Comparing with the result to the 1000ug/ml chart, we understood that we would need a higher concentration of Pe-4 to inhibit Botrytis cinerea.
Comparing the result to the 1000ug/ml chart, we understood that we would need a higher concentration to inhibit Botrytis cinerea.
Figure 15: 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 for Pe-4, the other peptides had a better effectiveness than our control.
If the percentage of ungerminated spores are more than the negative control, we defined the peptides under the concentration of 1000ug/mL as effective antifungal peptides.
In the premise of effective antifungal peptides, we then considered the concentration of 250ug/mL. We defined the ungerminated spores more than 50% of the spores as better antifungal peptides, which presented as circles in the table below; otherwise, they were the less effective peptides, presented in triangles.
For Pe-4, although the percentage of ungerminated spores at 1000 ug/mL was similar to that of the negative control, it was observed that Pe-4 was effective because the percentage of score_5 for Pe-4 was much less than the percentage of score_5 for the negative control.
Conclusion
With our experiment, we not only observed the suppression effect of each concentration of peptides but also the change in length of germ tubes in a micro view.
Table 12: The effectiveness of different peptides
Botany Experiment Result
Sample
Table 13: The sequences of peptides
Peptide vs. Concentration
Table 14: 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 pathogens and our peptide sprayed on the right.
Peptide Observation
Figure 16: Left: Botrytis cinerea infection on Oncidium petal; Right: uninfected Oncidium petal
- T-SN2
Figure 17: Left: Pathogens mixed with the negative control; Right: Pathogens 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 18: 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 19: 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 20: 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 21: 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 14: The result of different peptides
Final Result
Table 15: The figure shows the result of six peptides with three fungal experiments (circle: effective; triangle: less effective; X: ineffective).
We have conducted three experiments in order to validate our Parabase system. The spore germination experiment was the measurement for inhibiting spores, while the inhibition zone for inhibiting mycelia and the botany experiment for inhibiting the disease occurrence in reality.
There weren’t an absolute relationship among three experiments because they were conducted for different factors. As a result, we defined a peptide as an antifungal peptide if it has been proved to be antifungal in at least one experiment. Our overall result turned out to be successful, validating our Parabase as a high-level system.
Reference
[1]Antifungal Mechanism of a Novel Antifungal Protein from Pumpkin Rinds against Various Fungal Pathogens.J. Agric. Food Chem. 2009, 57, 9299–9304. DOI:10.1021/jf902005g
